The phenotype of CD3–CD56bright and CD3–CD56dim natural killer cells in systemic lupus erythematosus patients and its relation to disease activity

IntroductionSystemic lupus erythematosus (SLE) patients have decreased natural killer (NK) cell counts. The decrease in the number of NK cells has implications for a decrease in the function of NK cells which can affect the progression of SLE disease. The study aim was to determine profiles of CD3–CD56^bright and CD3–CD56^dim NK cells in SLE patients and their relation to disease activity.Material and methodsThis study included 36 patients of SLE who fulfilled the ACR 1997/SLICC 2012 criteria, women aged 18–49 years. Disease activity was assessed by the Mex-SLEDAI. Peripheral blood samples from SLE patients were analyzed by flow cytometry to evaluate NK cell subsets, according to differential expression of the main subset of NK cells, which is CD3–CD56^bright and CD3–CD56^dim.ResultsThe mean percentage of regulatory NK cell count (CD3–CD56^bright) in active SLE patients was significantly lower (p = 0.000) than in inactive SLE patients. The mean percentage of cytotoxic NK cell count (CD3–CD56^dim) in active SLE patients was significantly (p = 0.000) higher than in inactive SLE patients. A correlation was observed between two subsets of NK cells with disease activity (p = 0.00). The percentage of CD3–CD56^bright NK cells was negatively correlated with disease activity (r = –0.766), whereas the percentage of CD3–CD56^dim NK cells positively correlated with disease activity (r = 0.761).ConclusionsThere is a difference in the mean percentage of the number of NK cells (CD3–CD56+) in both a subset of regulatory NK cells (CD3–CD56^bright) and cytotoxic NK cells (CD3–CD56^dim) in active and inactive SLE patients and it is closely related to SLE disease activity.     

In-vitro effects of cyclosporin A, FK506, 6-mercaptopurine, and prednisolone on lymphokine-activated killer cells

In transplant recipients, immunosuppressive regimens are deleteriouson natural killer (NK) and lymphokine-activated killer (LAK)cells, which beyond their well-known antitumoral activity displaymany important biological functions. In order to find whichregimens could preserve NK and LAK functions we tested the influenceof CsA, FK506, 6-mercaptopurine and prednisolone on HLA-unrestrictedcytotoxicity during an in-vitro IL-2 activation. For each drug we obtained periphal blood samples from 11 healthyvolunteers. Non-adherent PBMC were incubated 2 days with eitherCsA, FK506, 6-mercaptopurine or prednisolone, whose concentrationsranged from 0 to 10 µg/ml, in order to screen infratherapeutic,therapeutic, and supratherapeutic doses. Thereafter, 100 IUof IL-2 were added for a further 3-day culture. Before and afterthe culture, we analysed (1) the cell subsets by direct immunofluorescence staining with anti-CD3/CD16/CD56 antibodies, (2) the LAKactivity with the lysis of Daudi cells, (3) the cell proliferationwith a 24-h incorporation of thymidine. Cyclosporin and FK506 did not impair the LAK cytotoxicity northe number of LAK cells, whereas both prednisolone and 6-mercaptopurinedecreased the LAK cytotoxicity, the number of CD3^– CD16⁺CD56⁺ cells, and the thymidine uptake. As a whole, the LAK cytotoxicitywas correlated with the number of CD3^– CD16⁺ CD56⁺ cellsbut not with the number of CD3^– CD16^– CD56^–cells, and it also increased with the incorporation of thymidine.This latter was correlated with the number of CD3^– CDl6⁺CD56⁺ cells, but not with the number of CD3⁺ CD16^– CD56^–cells. We conclude that in vitro the LAK activity is impaired by prednisoloneand 6-mercaptopurine, but not by CsA or FK506, and that thedeficiency of the LAK cytotoxicity seems to be related to thedecreased number of IL-2-activated NK cells.     

The induction of cytotoxicity by a bispecific antibody against CEA positive cell line,in vitro

A mouse anti-human carcinoembryonic antigen (CEA) × anti-human CD3 bispecific antibody, AB5C10*UCHT1, was developed. This antibody-heteroconjugate was chemically prepared by cross-linking the AB5C10 monoclonal antibody reactive with human CEA with the monoclonal antibody, UCHT1, which binds to CD3 on human T-lymphocytes. The AB5C10*UCHT1 recognized both CEA expressed on the KATOIII cell line and CD3 expressed on T-lymphocytes, as determined using flowcytometry. Next, AB5C10*UCHT1-mediated cytolysis was analyzed by ⁵¹Cr-release assay. When ⁵¹Cr-labeled target KATOIII cells were incubated for 6 h with effector cells that had been pretreated with AB5C10*UCHT1 for 60 min at 4°C, the percentage specific lysis was significantly increased compared to that of untreated effector cells. Using peripheral blood mononuclear cells (PBMC) and lymphokine-activated killer (LAK) cells pretreated with AB5C10*UCHT1 for effector cells, the percentage specific lysis was determined to be 16.3% and 57.4% at effector:target (E:T) ratios of 100:1 and 12.5:1, respectively. On the other hand, the percentage specific lysis of untreated PBMC and LAK cells determined to be 3.0% and 35.8% at E:T ratios of 100:1 and 12.5:1, respectively. The minimum effective dose of AB5C10*UCHT1 required for antibody-mediated cytotoxicity was 0.1 g/ml. The results of this study suggest that AB5C10*UCHT1 could be useful for augmenting the cytotoxicity of CD3-positive T-cells against CEA-positive target cells in vitro.     

自体LAK细胞和rIL－2治疗晚期肾癌的实验与临床研究

应用自体ＬＡＫ细胞和重组白细胞介素－２（ｒＩＬ－２）治疗２０例晚期肾癌患者。自患者周围血分离到的单个核细胞（ＰＢＭＣ）体外经ｒＩＬ－２短期培养，其ＮＫ、ＬＡＫ活性明显增强并于第５、７天达高峰。当这些ＬＡＫ细胞与ｒＩＬ－２过继回输给同一患者后，病人周围血ＮＫ、ＬＡＫ活性明显增加（Ｐ＜０．０ｌ），ＮＫ比率、ＩＬ－２受体表达明显增加（Ｐ＜０．０５），提示对肾癌患者的免疫调节作用。本组病人获部分缓解（ＰＲ）ｌ例，轻度缓解（ＭＲ）３例，平均缓解期５个月。毒副作用主要表现为发热、寒战，病人能耐受，表明ＬＡＫ／ｒＩＬ－２疗法是安全的方法。     

Characterization of baboon NK cells and their xenogeneic activity

Kennett SB, Porter CM, Horvath‐Arcidiacono JA, Bloom ET. Characterization of baboon NK cells and their xenogeneic activity. Xenotransplantation 2010; 17: 288–299. © 2010 John Wiley & Sons A/S. Abstract: Background: Baboons are commonly used as models for transplantation and preclinical testing of various types of therapeutic agents. For proper assessment of information gathered from these models, differences between the baboon and human immune systems need to be characterized. Natural killer (NK) cells are the first line of defense against many infectious agents and cancer and are important mediators of transplantation rejection reactions, particularly during xenotransplantation. In this study, we examined baboon NK cell function and developed methods for purifying and expanding these cells. Methods: Baboon NK cells were analyzed using a combination of extracellular and intracellular cell staining, cell sorting, interleukin (IL)‐2 mediated stimulation and expansion, and 4 h cytotoxicity assays with human and pig target cell lines. Results: Baboon peripheral blood mononuclear cell (PBMC) exert very low but detectable cytolytic activity against both human (K562) and pig (PAEC, J2) target cells, and this activity is enhanced within 4 h of treatment with IL‐2. Like human NK cells, many baboon PBMC express the lytic enzymes granzyme A, granzyme B, and perforin. Based on these markers, we identified a subpopulation of CD3^? baboon lymphocytes that are CD8^dim and CD16^bright that likely represents the baboon NK cells. These cells also are characterized by expression of the natural cytotoxicity receptor NKp46. Baboon CD3^?NKp46⁺ cells purified by flow cytometric cell sorting have high cytolytic capacity that can be further enhanced by IL‐2 stimulation. These baboon NK cells can be expanded in vitro and retain extremely high cytolytic capacity. While fresh baboon lymphocytes express very little CD56, the expanded baboon NK cells are predominantly CD56⁺; approximately 10% of the expanded NK cells are CD56^dim, and the remainder are CD56^bright. Conclusions: Baboon NK cells that are IL‐2 responsive can be identified on the basis of a CD3^?NKp46⁺CD8^dimCD16^+/? or CD3^?CD8^dimCD16^bright phenotype and can be isolated and expanded in culture. These results may allow for a more accurate representation of the human innate immune system in baboon models and more accurate analyses of the role of the baboon innate immune system cells in preclinical models.     

The interleukin-2 receptor α chain (CD25) plays an important role in regulating monocyte-derived CD40 expression during anti-porcine cellular responses

Long-term xenograft survival is limited by delayed xenograft rejection, and monocytes are thought to play an important role in this process. Although typically considered a T cell surface marker, interleukin 2 the receptor chain CD25 is also functional on monocytes. We hypothesized that CD25 expression on monocytes functions to augment monocyte activation in xeno-specific cellular responses. Xenogeneic mixed lymphocyte-endothelial cell reactions were used to study the role of CD25 in facilitating xenogeneic cell-mediated immune responses an in vitro. We also tested the effect of the anti-CD25 antibody daclizumab on monocyte-mediated T cell activation during xeno-specific cellular responses. Co-culture with porcine endothelial cells (PEC) elicited a pronounced proliferative response by human peripheral blood mononuclear cells (PBMC) that was accompanied by upregulation of CD25 and CD40 on CD14⁺ monocytes. CD4⁺ cells proliferated in response to PEC-conditioned monocytes, while blockade of CD25 with daclizumab reduced CD4⁺ cell proliferation in the presence of PEC-conditioned monocytes. In addition, daclizumab inhibited proliferation of PBMC in responses to PEC. Analysis of monocytes from PBMC-PEC cocultures by flow cytometry indicated that daclizumab inhibited CD40 upregulation on PEC-activated monocytes. These data demonstrate that CD25 blockade prevents xenogeneic cellular responses by directly blocking CD25 expression on both activated T cells and monocytes. CD25 blockade on T cells or monocytes may indirectly affect upregulation of CD40 on xenoreactive monocytes. Our data strengthen the rationale for incorporating CD25 directed therapy in discordant xenotransplantation.     

Analysis of T-cell receptor usage in myeloperoxidase−antineutrophil cytoplasmic antibody-associated renal vasculitis

Background

Bacterial superantigens produced by Staphylococcus aureus may be associated with the onset of proteinase-3 antineutrophil cytoplasmic antibody (PR3-ANCA)-associated vasculitis, including Wegener’s granulomatosis. We investigated T-cell subsets to assess the superantigens present in patients with myeloperoxidase?antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis.

Methods

Peripheral-blood mononuclear cells (PBMC) obtained from 40 normal controls and ten patients with MPO-ANCA-associated vasculitis were stained with fluorescence-labeled monoclonal antibodies against T-cell markers, including 17 variable regions of T-cell receptor β-chains (TCR-Vβ) and were then analyzed using flow cytometry.

Results

Among PBMCs, the percentage of CD3⁺ cells from patients with MPO-ANCA-associated vasculitis was significantly lower than that from normal controls, but there were no differences between the two groups in the percentage of CD19⁺ cells or CD16⁺ cells. Although there were no differences regarding the overall percentage of CD4⁺ cells between the two groups, the percentage of CD4⁺CD45RO⁺ cells in patients with MPO-ANCA-associated vasculitis was significantly higher than that in normal controls, and percentages of CD4⁺CD45RO⁺HLA-DR⁺ and CD4⁺CD45RO⁺CD62L^low cells in patients with MPO-ANCA-associated vasculitis were also significantly increased. There was no significant difference between the two groups in terms of the usage of the 17 different TCR-Vβ regions.

Conclusion

There was no difference in bacterial superantigens between controls and MPO-ANCA-associated vasculitis patients because of the absence of specific usage of TCR-Vβ regions. Given the elevated levels of memory T cells, conventional antigens rather than superantigens may be associated with the pathogenesis of MPO-ANCA-associated vasculitis.     

Rabbit anti-human leukocyte polyclonal antibody inhibits xenogeneic cell-mediated immune responses

We studied the interactions between human monocytes and porcine endothelial cells (PEC) as well as the effects of a new generation of rabbit anti-human leukocyte polyclonal antibody (newRALG) to inhibit xenogeneic cell-mediated immune responses. Human peripheral blood mononuclear cells (PBMC) were cocultured with the florescent dye PKH-26 labeled-PEC, which showed membrane uptake by monocytes detected by florescence activated cell scanning (FACS). Scavenger receptor (SR) ligand poly-(G) or the newRALG or Thymoglobulin was added into the cocultures followed by FACS. Lymphocyte proliferation upon exposure to PEC with or without newRALG or thymoglobulin was evaluated by a xenogeneic mixed-lymphocyte-endothelial cell reaction (xMLER). FACS analysis demonstrated that CD14⁺ monocytes became positive for PKH-26 following their interaction with PKH-26-labeled PEC. These PKH-26⁺ monocytes displayed up-regulated CD40 and CD80 expression during the PBMC-PEC interaction. Furthermore, SR blockade with poly-(G) prevented PEC membrane uptake by CD14⁺ monocytes. The newRALG from rabbits immunized with activated human monocytes and lymphocytes greatly reduced SR-mediated PEC membrane uptake. xMLER demonstrated strong lymphocyte proliferation in response to PEC. Lymphocyte proliferation was dramatically inhibited in dose-dependent manner by the newRALG. In summary, monocytes up-regulate costimulatory molecules during xenogeneic interactions, indicating that they may serve as a source of T-cell costimulation during xenogeneic reactions, enguling PEC membranes. This phenomenon was inhibited by poly (G), suggesting that PEC membrane uptake was via SR. The newRALG inhibited monocyte SR-mediated PEC membrane uptake and lymphocyte proliferation in response to PEC suggesting that this new polyclonal preparation may impair the initiation of xeno-specific immune responses.     

Assessment of in vitro lymphokine activated killer (LAK) cell activity against renal cancer cell lines and its suppression by serum factor using crystal violet assay

Summary Lymphokine activated killer (LAK) cell activity against renal cancer cell lines was assessed in vitro using a crystal violet assay. A standard 4-h ⁵¹chromium release assay and a 48-h crystal violet assay showed that both natural killer-susceptible (NC65) and-resistant (ACHN) renal cancer cell lines were sensitive to LAK cells which had been generated by a 3-day incubation of peripheral blood mononuclear cells (PBMC) with recombinant interleukin 2 (rIL-2). Optimal LAK activity was generated by a 5-day culture of PBMC with 1 U rIL-2/ml. LAK activity was enhanced by the presence of IL-2 in the crystal violet assay system, while it was suppressed by fresh autologous serum. The suppressive effect was found in serum from both normal donors and patients with metastatic renal cell carcinoma, suggesting that non-specific suppressive factor(s) affecting LAK cell activity were present in human sera.     

Foxp3+ T cells in peripheral blood of renal transplant recipients and clinical correlations

Aim: Immunophenotype peripheral blood T cells from renal transplant recipients (RTR) using cellular markers of regulatory T cells (Tregs) and flow cytometry, including Foxp3, and correlate these findings with clinical parameters. Methods: Expression of phenotypic markers of Tregs was assessed by flow cytometric analysis of peripheral blood lymphocytes (PBL) from (i) RTR (n = 95); (ii) patients with end‐stage renal failure (ESRF) awaiting transplantation (n = 17); and (iii) normal healthy controls (n = 18). Results: The percentage of CD4⁺CD25⁺Foxp3⁺ cells within the CD4⁺ cell population did not significantly alter at different time points post‐transplant. However, the percentage of CD4⁺CD25⁺Foxp3⁺ cells within the CD4⁺ population was significantly lower in RTR compared with patients with ESRF. In contrast, RTR and ESRF had a similar percentage of CD4⁺CD25⁺ cells expressing Foxp3. Multivariate analysis of PBL and clinical parameters demonstrated (i) a positive linear relationship between the percentage CD4⁺CD25⁺ cells expressing Foxp3 and estimated glomerular filtration rate and (ii) a higher percentage of CD4⁺CD25⁺ cells in the CD4⁺ cell population in patients with malignancy (the majority were skin cancers). Malignancy also correlated strongly with time post‐transplant and age of the RTR. Conclusion: Immune monitoring of the PBL phenotype in RTR using CD4, CD25 and Foxp3 may stratify RTR and predict graft outcome and function, and risk of complications from immunosuppression. Longitudinal and functional studies of Tregs are essential to extend the findings of the present study.     

Influence of multimodality therapy on the cellular immunity of patients with esophageal cancer

Background: Cancer patients have often been reported to have impaired immune function, and the effect of treatment modalities, such as surgery, irradiation, and chemotherapy, in depressing patients’ immunity has also been reported. In this investigation, the effect of treatment on the cellular immunity of esophageal cancer patients was evaluated. Methods: Immunological parameters, such as natural killer (NK) activity and lymphocyte subsets in peripheral blood, were measured in 32 esophageal cancer patients on 5 occasions (on the day of admission, 2 days before surgery, and 1 week, 1 month, and 2 months after surgery). Results: NK activity was greatly impaired shortly after the operation, and the percentages of lymphocytes as a whole, and CD8⁺, CD16⁺, and CD57⁺ lymphocytes were significantly decreased, on the other hand, a postoperative increase in the CD4⁺/CD8⁺ ratio was observed. No significant depression of immune function by postoperative irradiation was observed. Conclusions: The results of this study suggest that cellular immunity, especially cytotoxicity, shortly after esophagectomy may be greatly impaired by the surgical stress of esophagectomy and an added effect of chemotherapy.     

The percentage of CD31<Superscript>+</Superscript> T cells decreases after open but not laparoscopic surgery

Background: Efficient killing of tumor cells depends on T cells that migrate from the circulation to the peripheral tissues; these cells express CD31. This study was undertaken to determine the impact of open (OS) and laparoscopic (LS) colorectal surgery on the percentage of circulating CD3⁺CD31⁺ cells. Methods: Peripheral blood was collected from 27 OS and 24 LS colon cancer patients preoperatively (preOP) and on postoperative days 1 (POD1) and 3 (POD3). CD31⁺ T cells were assessed by flow cytometry using monoclonal antibodies. Results: In the OS group, the percentage of CD3⁺CD31⁺ cells was significantly lower in POD1 and POD3 samples compared to the preOP results. LS surgery did not result in a significant change in the percentage of these T cells. A significant correlation was found between the decrease in the percentage of CD3⁺CD31⁺ cells and the length of incision in OS patients. Conclusions: The percentage of CD3⁺CD31⁺ cells decreases following OS but not LS and may be related to incision length. This may compromise T cell function in the peripheral tissues in the postoperative period.     

Decreased levels of perforin-positive lymphocytes are associated with posttraumatic complications in patients with major trauma

ObjectivePosttraumatic immune disorder can cause complications including systemic inflammatory response syndrome (SIRS) and multiple-organ dysfunction syndrome (MODS). Cytotoxic granules containing perforin and granzyme-B (GrB) are released by cytotoxic CD8⁺ T lymphocytes, NK and γδT cells after major trauma. This prospective clinical study was designed to analyze the association between these immune components and complications after major trauma.MethodsWe retrospectively studied 48 patients aged between 16 and 65 years admitted within 90 min of major trauma (Injury Severity Score > 16) and surviving beyond 7 days, and 20 healthy controls. Blood samples were drawn on admission and after 1, 3 and 7 days. CD8⁺ T, NK and γδT cell counts in peripheral blood and the levels of perforin and GrB in these cells were analyzed by flow cytometry. Clinical aspects of MODS and SIRS were recorded daily.ResultsCD8⁺ T cell counts were not significantly different in patients with SIRS or uncomplicated group, but were depressed in the MODS group after trauma. However, NK cell counts in patients with MODS were significantly depressed only at day 7 after injury, and γδT cell counts were significantly depressed after trauma. Perforin levels in CD8⁺ T, NK and γδT cells in patients with MODS were depressed after trauma. GrB levels in NK, CD8⁺ T and γδT cells in patients with MODS were significantly depressed at 3 and 7 days post trauma.ConclusionPosttraumatic MODS is associated with early, sustained, and severe depression of lymphocytes.     

Mechanisms of enhanced osteoclastogenesis in girls and young women with Turner's Syndrome

Subjects with hypergonadotropic hypogonadism due to Turner's syndrome show low cortical mineral density, osteoporosis and risk of fractures. It is not clear if this bone fragility derives from chromosomal abnormalities or is the result of inadequate bone formation due to estrogen deficiency.The aim of this study was to investigate the cellular mechanisms underlying bone fragility in subjects with Turner's syndrome before induction of puberty and after hormonal replacement therapy (HRT). For this purpose, we have evaluated the osteoclastogenic potential of non-fractioned and T-cell depleted cultures of peripheral blood mononuclear cells (PBMCs) belonging to girls with Turner's syndrome who had not been treated with HRT yet, girls and young women who were on HRT and age-matched controls. Untreated subjects showed high FSH serum levels, whereas the other subjects displayed normal FSH serum levels. T-cell immunophenotype was analyzed through flow cytometry. Biochemical and DXA analyses were performed.Spontaneous osteoclastogenesis in non-fractioned and T-cell depleted cultures of PBMC belonging to girls with high FSH levels was more evident than in cultures of subjects with normal FSH levels. In the former, osteoclastogenesis was sustained by monocytes expressing high levels of c-fms, TNF-α and RANK, and T-cells producing high RANKL and TNF-α; in the latter it was supported by T-cells expressing high RANKL levels. CD4⁺CD25^high T-cells were reduced in all subjects, whereas CD3⁺/CD16⁺/CD56⁺ NKT-cells were increased in those with high FSH levels. High RANKL and CTX levels were detected in the sera. Bone impairment was already detectable by DXA in subjects aged under 10, although it became more evident with aging.In conclusion, our results demonstrated that bone fragility in subjects with Turner's syndrome is associated to enhanced osteoclastogenesis. This process seems to be due to high FSH serum levels before HRT, whereas it is caused by high RANKL during HRT.     

Immune phenotype of peripheral blood mononuclear cells in patients with high-risk non-muscle invasive bladder cancer

Purpose

To explore the immune phenotype of peripheral blood mononuclear cells (PBMC) in patients with high-risk non-muscle invasive bladder cancer (NMIBC).

Methods

We prospectively collected blood samples from patients with high-risk NMIBC treated at our institution. PBMC were analyzed by flow cytometry to determine the frequency of T cells and NK cells and the expression of immunoregulatory molecules (Tim-3, TIGIT and PD-1). PBMC from healthy donors (HD) were included for comparison, and associations with response to BCG were investigated.

Results

A total of 38 patients were included, 19 BCG responders and 19 BCG refractory. Compared to 16 PBMC from HD, the frequency of total NK cells was significantly higher in patients with NMIBC [15.2% (IQR: 11.4, 22.2) vs. 5.72% (IQR: 4.84, 9.79); p?=?0.05], whereas the frequency of T cells was not statistically different. Both Tim-3 and TIGIT expressions were significantly higher in NMIBC compared to HD, particularly in NK cells [13.8% (11.0; 22.4) vs. 5.56% (4.20; 10.2) and 34.9% (18.9; 53.5) vs. 1.82% (0.63; 5.16), respectively; p?<?0.001]. Overall, the expression of PD-1 in all cell types was low in both NMIBC patients and HD. The immune phenotype was not significantly different before and after initiation of BCG. However, the proportion of CD8⁺ T cells before BCG was significantly higher in responders.

Conclusion

The immune phenotype of PBMC from patients with high-risk NMIBC was significantly different from HD, regardless of the presence of disease or the initiation of BCG. Peripheral CD8⁺ T cells could play a role in response to BCG.

     

Low expression of glucocorticoid receptors in children with steroid-resistant nephrotic syndrome

Background

About 10–20 % of children with idiopathic nephrotic syndrome (NS) are steroid-resistant (SR). Low expression of glucocorticoid receptors (GRs) has been associated with poor response to steroids in a variety of autoimmune diseases. This study was done to assess the expression of cytoplasmic GRs for CD3 and CD14 in children with NS.

Methods

Expression of cytoplasmic GRs in lymphocytes (CD3⁺/GR) and monocytes (CD14⁺/GR) in the peripheral blood were assessed in 51 children with NS before the start of therapy by flow cytometry. Patients were divided into two groups: 30 children who were steroid-sensitive (SSNS) and 21 children who had initial steroid resistance (SRNS). Twenty age- and sex-matched healthy children served as controls.

Results

Expression of CD3⁺/GR was significantly lower in SRNS in comparison to SSNS patients and controls (p?<?0.0001). Similarly, expression of CD14⁺/GR was significantly lower in SRNS in comparison to SSNS patients (p?<?0.0001) and controls (p?=?0.002). CD3⁺/GR and CD14⁺/GR expression were not significantly different in SSNS patients compared with controls (p?=?0.06 and 0.07 respectively).

Conclusions

Patients with initial SRNS showed decreased GR expression in peripheral blood mononuclear cells (PBMC) before starting therapy, and this low expression may be one of the pathophysiological mechanisms of steroid resistance in these children.     

Investigating the relationship between the number and activity of natural killer cells with increased cytomegalovirus and CMV disease after kidney transplantation

BackgroundCytomegalovirus (CMV) infections caused by the cytomegalovirus are one of the most common problems in patients after kidney transplant. We examined the association of the relationship between the number and activity of natural killer cells with increased cytomegalovirus and its related disease after kidney transplantation.Material and methodsIn this analytical study, 58 new transplant patients in the Labbafinejad Hospital, who did not have any evidence of CMV infection, were evaluated based on the number and percentage of CD56⁺/16⁺, CD56⁺/16⁻, and CD69⁺ Natural Killer (NK) cells.ResultsThe results of this study showed that CD16⁺ and CD56⁺ cells in the group of CMV Ag-positive patients are less than negative patients (p = 0.003) and the difference between the two groups are significant (p = 0.01). However, CD69⁺ cells did not differ significantly between the two groups (p = 0.1). Moreover, the absolute number of CD16⁺ and CD56⁺ cells declined significantly after infection with CMV unlike the CMV Ag - group(p = 0.003).DiscussionThese results indicate that kidney transplant patients suffering from CMV infection after transplantation have a significantly reduced total number of NK cells. On the other hand, a slight decrease in the number of NK subgroups was observed with an increase in the peak serum levels of cyclosporine. As a consequence of these findings, it can be assumed that more dosage and a higher level of the drug will result in more severe immunosuppression and, consequently, increased susceptibility to CMV infections. Thus, taking the right dose of the drug would prevent viral infections and immune system from over-activation.     

Cytokine production and cytotoxicity of lymphocytes in patients on maintenance short- or long-term haemodialysis

The lymphocyte-mediated cytotoxicity and cytokine productionof patients under maintenance haemodialysis were studied. Thepatients were subdivided into two groups; 27 patients receivinghaemodialysis treatment for up to 3 years (group 1) and 27 patientsfor longer than 10 years (group 2). Twenty-six healthy volunteers(group 3) were used as controls. The immunological parametersassayed were NK cell and lymphokine-activated killer (LAK) cellactivities, and production of both interferon (IFN)-gamma andtumour necrosis factor (TNF)-alpha by either peripheral bloodmononuclear cells (PBMC) cultured with IL-2 or LAK cells. In group 1 both NK- and LAK-cell-mediated cyto-toxicities weresignificantly suppressed as compared to those in group 3. Thetitre of IFN produced by LAK cells was also less than that ingroup 3. On the contrary there were no significant differencesin cytokine production by PBMC in all three groups. Percentageof the cases with suppressed cytokine production seemed higherin group 1 than group 3. In addition there was a close correlationbetween the value of IFN-gamma and of TNF-alpha produced byPBMC in the patients on haemodialysis. Both the type of dialysismembranes used and the primary diseases did not significantlyinfluence the results in group 1 and group 2. From these resultsit could be speculated that there are more patients with impairedimmune function in group 1 than in group 3. In contrast therewas no significant difference in the immune function testedbetween group 2 and group 3. Our results indicate the need for a study on time kinetics concerningthe change in suppressed immune function of the patients andtheir clinical courses shortly after the beginning of haemodialysis.     

Prognostic value of the immunomonitoring of patients with renal cell carcinoma under therapy with IL-2/IFN-α-2 in combination with 5-FU

After tumor nephrectomy, patients suffering from metastatic renal cell carcinoma (RCC) received interleukin-2 (IL-2), interferon (IFN)--2b and 5-fluorouracil (5-FU) in one to three treatment cycles over 8 weeks Using flow cytometry, we investigated the immunophenotype of peripheral blood lymphocytes from 22 patients during therapy. In all patients, we found an increase in the absolute number of T lymphocytes, especially of the CD4 type, and in the number of HLA-DR⁺, CD25⁺ T cells and natural killer (NK) cells. The mean number of B cells did not increase during therapy. The numbers of CD4⁺, CD8⁺ and CD25⁺ T cells correlated significantly with the clinical response. In addition, we found that the pretherapeutic number of T lymphocytes and B cells but not of NK cells was significantly higher in patients with a therapy-induced clinical response. In conclusion, we describe the predictive value of the number of lymphocytes from peripheral blood for the efficiency of IL-2/IFN--2b therapy in combination with 5-FU in patients with metastatic renal cell carcinoma.     

Calcitriol effect on natural killer cells from hemodialyzed and normal subjects

Patients with chronic renal failure have a decreased secretion of calcitriol (CTR). They also show an impaired cellular immune response including a defective natural killer (NK) cell-mediated activity. The aim of this study was to analyze, in vivo and in vitro, the effect of CTR on NK cell cytotoxicity in healthy control subjects and in hemodialyzed (HD) patients. Our results show that HD patients had baseline-depressed NK cell activity when compared with controls (P<0.001), which increased significantly after 1 month of oral CTR treatment (0.5 g/day) (P<0.001). Calcitriol treatment also induced a significant increase in CTR serum levels (P<0.001) and a significant decrease (P<0.001) in total parathyroid hormone (PTH). In vitro CTR treatment (10^-7 M) of peripheral blood mononuclear cells (PBMC) increased NK cell-mediated cytotoxicity after 24 hours of incubation with a maximum at 48 hours (P<0.001). In vitro CTR treatment at doses of 10^-11 and 10^-9 M did not significantly increase NK cytotoxic activity. The enhanced NK activity after CTR treatment was not the consequence of increased numbers of CD56 positive cells, nor to lymphocyte activation, as tested by the expression of the interleukin 2 receptor p55 chain (CD25) on their surface. In vitro treatment of PBMC from HD patients with CTR (10^-7 M, during 48 hours) also induced a strong increase in NK cell cytotoxicity (P<0.001). These results demonstrate a positive role of CTR in the stimulation of NK cell activity and support the hypothesis of a direct steroid-mediated action of CTR on NK cells, although an indirect effect mediated by the CTR-induced PTH decrease in vivo cannot be excluded. Our data also raise the possibility for potential therapeutic uses of this hormone in immunomodulation of patients with depressed NK cell activity.