Peripheral Lymphocytes Of Patients Research Articles

Infantile rickets reduces lymphocyte survival

Increased incidence of infections had long been recognized in rachitic patients who were also described to have impaired lymphocytic functions. This study was designed to assess different apoptotic changes in peripheral lymphocytes in patients with infantile rickets in its various stages and to correlate these findings with the frequency of infection in this disease. The study included 24 rachitic patients with a mean age of 17.88 ± 7.65 months compared with 16 healthy matching controls. There were 7 rachitic patients who were in the active stage, 11 in the healing stage, and 6 in the healed stage. The enrolled cases were subjected to a full history and clinical examination. X-ray of extremities was performed, in addition to the laboratory tests including serum calcium, phosphorus, and alkaline phosphatase as well as direct immunofluorescence using flow cytometry with dual staining technique to check for apoptotic changes in peripheral lymphocytes. The results of the present study showed that the early apoptotic cells percent (EAC%), the late apoptotic and/or necrotic cells percent (LA and/or NC%), the NC%, and the total abnormal cells (%) of the lymphocytes were higher in the rachitic patients compared with healthy controls; although only the latter showed statistical significance. The EAC% of lymphocytes showed a trend to increase accompanied by a simultaneous decrease in the LA and/or NC% with the progression toward the healed rickets stage. The current study also showed a trend of the EAC% and LA and/or NC% of the lymphocytes to decrease with the doses of vitamin D shock therapy given to the rachitic patients. A significant positive correlation was demonstrated between the EAC% and the total abnormal cells (%) of the peripheral lymphocytes and the rate of chest infection in the rachitic cases in the 3 months previous to the study. In conclusion, rickets does not increase the rate of infection through local causes or immune cell dysfunction only, but it entails disturbed lymphocyte survival. The increased apoptotic tendency of peripheral lymphocytes in rickets is reversed with vitamin D therapy, which further emphasizes the importance of early prevention and proper treatment of such a disease.

Multi-drug resistance in the treatments of autoimmune diseases

Although corticosteroids and immunosuppressants are widely used for the treatments of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), we often experience patients who are resistant to these treatments. P-glycoprotein (P-gp) of membrane transporters, a product of the multiple drug resistance (MDR)-1 gene, plays a pivotal role in the acquisition of drug resistance. However, the relevance of MDR-1 and P-gp to resting and activated lymphocyte, major targets of the treatments in autoimmune diseases, remains unclear. We found that peripheral lymphocytes in patients with SLE and RA express P-gp on the surface and its expression is highly correlated with disease activity. P-gp on lymphocytes is induced by activation with cytokines such as IL-2, IL-4 and TNF-alpha, resulting in active efflux of corticosteroids from cytoplasm of lymphocytes, which mechanisms could lead to drug-resistance and high disease activity. However, the addition of P-gp antagonists such as ciclosporin A and inhibitors of P-gp synthesis successfully reduce efflux of corticosteroids from lymphocytes in vitro and these results imply that P-gp antagonists and P-gp synthesis inhibitors could work in order to overcome drug-resistance in vivo. Therefore, we propose that measurement of P-gp on lymphocytes is useful marker to indicate drug resistance and requirement of antagonists and/or intensive treatments to overcome drug resistance in active SLE and RA patients.

Telomerase reverse transcriptase mRNA expression in peripheral lymphocytes of patients with chronic HBV and HCV infections

Telomerase activity is present at low levels in peripheral lymphocytes (PL) and is upregulated upon activation, possibly protecting PL from telomere shortening. As decreased telomere length is considered a sign of cellular senescence, telomerase may, therefore, play an important role on immune function, organ regeneration and carcinogenesis. So far, quantification of human telomerase reverse transcriptase levels (hTERT) in PL, has not been reported. We determined hTERT mRNA levels in PL of hepatitis B virus (HBV) and hepatitis C virus (HCV) patients, in an attempt to address whether hTERT transcripts in PL are altered in these viral diseases, which are characterized by immune dysfunction and increased incidence of hepatocarcinogenesis. hTERT mRNA levels in PL of HBV (n = 17), HCV (n = 24) patients and healthy controls (n = 22) were quantified by real-time polymerase chain reaction. We observed significantly lower hTERT mRNA levels in HBV and HCV patients compared with healthy individuals (P < 0.05). hTERT mRNA levels were not associated with the patients' clinical status (inactive, hepatitis and cirrhosis). Also no correlation was observed between hTERT mRNA expression, and HBV and HCV replicative activity. In the inactive group (n = 18) we observed a negative correlation between hTERT mRNA expression and disease duration (rs = -0.52, P < 0.03). We performed for the first time an accurate quantification of hTERT mRNA expression in PL of HBV and HCV patients. The observed low levels of hTERT mRNA expression in the above patients may suggest its involvement in the immunopathogenesis of chronic viral hepatitis.

Genotoxicity and sister chromatid exchange in patients with myelodysplastic disorders

In this study, we aimed to evaluate genotoxicity by sister chromatid exchange frequency in the peripheral lymphocytes of patients with myelodysplastic syndrome (MDS). The study population consisted of 16 patients (females/males:6/10; mean age: 61.68 ± 13.08 years) diagnosed with “refractory anemia” according to FAB classification. The results were compared with an age- and sex-matched control group. The sister chromatid exchange frequency in the study group was found to be significantly higher than the control group (8.3 ± 11 vs. 6.83 ± 1.07, P = 0.0046). We suggest that increased DNA damage, which is revealed by the increased sister chromatid exchange in the present study may play a role in the etiopathogenesis of MDS.

Lack of Association of Common Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutations with Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC) is a chronic progressive cholestatic liver disease of uncertain etiology. However, the histologic features of PSC liver disease can resemble those in cystic fibrosis (CF), an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to determine if PSC patients have a higher frequency of common CF alleles than disease controls. DNA was extracted from peripheral lymphocytes of patients with end-stage liver disease. Samples were obtained before liver transplantation from 59 PSC patients and from three groups of control patients (20 each with primary biliary cirrhosis, autoimmune hepatitis, or hepatitis C). DNA samples were genotyped for 32 common CF mutations, the intron 8 T tract variants, and the M470V variant. One of 59 PSC patients (1.7%) had the common CF mutation (DeltaF508) in one CFTR gene. Two controls (3.3%) carried a single CF mutation (DeltaF508 in one primary biliary cirrhosis patient; W1282X in one hepatitis C patient). These rates do not differ from expected in the general population. The frequency of CFTR variants (5T and M470V) was also similar between PSC patients and controls. Despite anatomical similarities between CF liver disease and PSC, we could not confirm that PSC patients carried common CF mutations or common CFTR variants in higher than expected frequencies. These data suggest that CFTR dysfunction does not influence the pathogenesis of PSC.

Impeded Th1 CD4 memory T cell generation in chronic-persisting liver infection with Echinococcus multilocularis

Memory T cells of the CD4 lineage coordinate immune responses against pathogens via the antigen-induced secretion of potent effector cytokines. The efficacy of these responses is thought to depend on both the overall number of pathogen-specific memory T cells and the particular array of cytokines that these cells are programmed to secrete. It is unknown to what extent cellular immunity can be induced by Echinococcus multilocularis infection. To examine the immunological memory provided by the adaptive cellular immune system in control of the chronic-persisting infection, peripheral lymphocytes of patients with alveolar echinococcosis (AE) were studied ex vivo. Stimulation of memory cells was performed with E. multilocularis vesicular fluid, purified protein derivative as recall antigen and phytohemagglutinin. Cytomegalovirus latency served as disease control. Frequencies of circulating CD4(+) T cells secreting IFN-gamma, IL-2, tumor necrosis factor-alpha, IL-4, IL-5 and IL-10 were determined by both cytokine flow cytometry and ELISPOT assays. Most strikingly, in chronic AE the frequencies of E. multilocularis antigen-specific cells committed to T(h)1-cytokine production were low (mean 0.5% of CD4(+) T cells). However, an E. multilocularis-specific response of CD4(+) T cells at frequencies of >/=0.1% was detected in the majority of AE patients (68%). Low numbers of cells committed to T(h)1 cytokine secretion were invariably seen in patients with active and inactive disease. Interestingly, the number of specific CD4(+) memory T cells was not increased in cured AE patients after complete surgical removal of the metacestode. Hyporesponsiveness during the chronic helminth infection was E. multilocularis specific. Thus, our results demonstrate that antigen-specific memory function against E. multilocularis is markedly different from that against viral or bacterial pathogens. Whether the antigen-specific cellular hyporesponsiveness with impeded T(h)1 CD4(+) memory T cell generation is a cause or a result of the progressive metacestode activity remains to be determined.

Expression of HSP70 in peripheral lymphocytes of the patients with allergic rhinitis.

The expression levels of heat shock protein 70 (HSP70) from peripheral lymphocytes of the patients with allergic rhinitis (AR) and the clinical implication were investigated. In the morning, 3 ml of fasting venous blood was taken out. The lymphocytes were isolated by using Ficoll-Hypaque and the expression of HSP70 in the lymphocytes was detected by using Western blot. In the AR patients the HSP70 level (41.49 +/- 15.77 integrated optical density, IOD) were significantly higher than that in the control group (23.89 +/- 10.13 IOD, P < 0.05). Western blot demonstrated that HSP70 bands in AR patients were more intensive than those in the control group. It was concluded that the elevated HSP70 level in peripheral lymphocytes of the AR patients might contribute to the development of AR.

122 Increased production of tumour necrosis factor-alpha in peripheral lymphocytes in patients with heart failure

122 Increased production of tumour necrosis factor-alpha in peripheral lymphocytes in patients with heart failure

An analysis of DNA repair as a determinant of survival in patients with non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is frequently resistant to chemotherapy, and this resistance has been associated with elevated nucleotide excision repair (NER) in tumor tissue. We hypothesized that patients with NSCLC who had effective systemic (host) NER would have poorer survival than patients with suboptimal NER and that the association between NER effectiveness and survival would be most marked in patients receiving chemotherapy. 375 patients with newly diagnosed NSCLC were accrued for a case-control study between July 1995 and December 1999. NER activity was estimated as the DNA repair capacity (DRC) measured in the patient's peripheral lymphocytes by the host cell reactivation assay. Cox proportional hazards models were used to assess the association between DRC and survival. All statistical tests were two-sided. For every unit (percentage) increase in DRC, the relative risk (RR) of death was 1.05 (95% confidence interval [CI] = 1.00 to 1.10; P =.05) for the 345 patients for whom weight loss information was available and 1.06 (95% CI = 1.00 to 1.12; P =.03) for the 275 patients with complete follow-up information. In 86 patients treated with chemotherapy only, the RR of death increased to 1.11 (95% CI = 1.02 to 1.21; P =.01) for every unit (percentage) increase in DRC. Of those 86 patients, patients in the top quartile of the DRC distribution were at twice the RR of death as those in the lowest quartile (RR = 2.72; 95% CI = 1.24 to 5.95; P =.01). Effective DRC was not a risk factor for death in patients who were not treated with chemotherapy. Our data suggest that effective host DRC may be associated with poorer survival in patients with NSCLC who are treated with chemotherapy.

Increase in peripheral CD4 bright+ CD8 dull+ T cells in Parkinson disease.

Immune abnormalities are known to be involved in the pathogenesis of sporadic Parkinson disease. To examine whether abnormalities in peripheral lymphocytes exist in Parkinson disease. Immune mediators, including CD1a, CD3, CD4, CD8, CD45RO, and Fas (CD95), were examined in peripheral lymphocytes of patients by 3-color flow cytometry. Patients with Parkinson disease displayed a significantly greater population of circulating CD3+ CD4 bright+ CD8 dull+ lymphocytes than age-matched control subjects (P =.005) and patients with cerebrovascular disease (P =.002). The increase in these cells appeared to continue for at least 17 months. These T cells also expressed CD45RO and Fas, markers for activated T cells, while CD1a, a marker for thymic T cells, was negative, suggesting that these cells are mature T cells with immune activities. As CD4+ CD8+ T cells are known to increase after some specific viral infections, the continuous increase in CD4 bright+ CD8 dull+ T cells shown here may indicate postinfectious immune abnormalities that are possibly associated with the pathogenesis of this slowly progressive, multifactorial neurodegenerative disease.

Peptide-specific cytotoxicity of T lymphocytes against glutamic acid decarboxylase and insulin in type 1 diabetes mellitus

Cytotoxic T lymphocytes (CTL) against pancreatic β-cells probably play a major role in the etiology of type 1 diabetes mellitus (DM). CTLs recognize a complex formed between MHC class I and antigenic peptides fragments derived from intracellular processing of proteins. However, the exogenous peptides, which show strong affinities to MHC class I, can be presented. In this study, we focused on the cytotoxic activity of peripheral lymphocytes in patients with type 1 DM against the peptides of glutamic acid decarboxylase (GAD) and insulin, which can bind MHC class 1 A24. Lymphocytes were isolated from peripheral blood of 12 type 1 DM patients and eight healthy control subjects. The effector cells were cultured with peptides, IL-2 and IL-7, restimulated weekly by autologous antigen presenting cells, which were cultured with IL-4 and GM-CSF. On day 21, CTL activities of cultured effector cells were tested against autologous EB-blast cells as target cells pulsed with the stimulating peptides using 51Cr release assay. The results showed that cytotoxicity against insulin peptide binding to MHC class I A24 was observed in lymphocytes of four out of ten patients with type 1 DM. The mean cytotoxicity was 46.0% of the maximum release. The antibody against HLA-class I inhibited this effect. Cytotoxicity against GAD peptide which bind MHC class I A24 was not observed in seven patients. None of healthy controls showed cytotoxicity against GAD or insulin peptides was observed. This is the first report describing the cytotoxic activity of CD8 + T lymphocytes against insulin in type 1 DM.

Interferon-&amp;ggr' production in peripheral lymphocytes of patients with tinea pedis: comparison of patients with and without tinea unguium

Interferon-&amp;ggr' production in peripheral lymphocytes of patients with tinea pedis: comparison of patients with and without tinea unguium

Sister chromatid exchange in patients with joint prostheses

The evaluation of sister chromatid exchanges (SCE) is used to establish the cytogenic damage in subjects exposed to toxic substances. The test is considered to be 1 of the most sensitive and accurate indicators of damage and responds to toxic chemicals at low doses. We evaluated the incidence of SCE in peripheral lymphocytes of patients with articular prostheses. Subjects with prostheses made of titanium-aluminium-vanadium alloys presented a significantly higher SCE number than the control population (6.3±2.3 vs 4.4±1.3; P =.0128), whereas subjects with prostheses made of chrome-cobalt alloy or mixed prostheses presented a higher SCE value than the controls but not significantly different. The presence of high-frequency cells was alarming only in 5 patients, 4 of whom had titanium alloy prostheses, whereas none belonged to the control group. The number of SCE was not affected by the presence of bone-cement used in prosthesis fixation or by the implant duration. The indication of possible cytogenic damage in patients with titanium alloy prostheses that emerged from this study should be considered carefully, even though the sample population was small.

Lymphocyte subpopulations in patients with advanced breast cancer submitted to neoadjuvant chemotherapy.

There is an enhanced immune response in patients with breast cancer after the use of chemotherapy. The objective of this study was therefore to investigate alterations in the number of peripheral lymphocytes in patients with breast cancer after neoadjuvant chemotherapy (NC) and the relationship with prognosis. Thirty women were analyzed. Their UICC staging was IIb (only T3N0 included) and III (N3 not included). Sample analysis was performed using flow cytometry before the first cycle and 18 to 21 days after the last cycle of NC. The lymphocyte subsets studied were: T (CD3, CD4, CD8), B (CD19, CD23), natural killer (NK) (CD56, CD16), and interleukin-2 (CD25). CD3, CD56, CD8, and CD16 lymphocytes were analyzed with double marking. After x = 3.8 +/- 1.3 cycles of 5-fluorouracil, epirubicin and cyclophosphamide (FEC), 16 patients showed a complete or partial response (group 1). After three cycles 14 showed no response or tumor progression (group 2). A control group of healthy women was used for pretreatment analysis. Before NC there was a significant increase in B lymphocytes and NK cells in comparison to the control group. After NC there was a significant percentage increase in CD3, CD4, CD8, CD25 and CD3+CD56+ cells and a decrease in CD19, CD23, CD56, CD16 and CD16+CD8+ cells. There was a significant fall in the absolute number of CD4, CD19, CD23, CD56, CD16 and CD16+CD8+ lymphocytes and an increase in CD3+CD56+ lymphocytes. Before NC the ratio CD4/CD8 in group 1 was 2.25 +/- 0.5 and in group 2 it was 1.79 +/- 0.5 (P <0.05). Patients with advanced breast cancer showed increases in B and NK lymphocytes. Neoadjuvant chemotherapy (FEC) caused an increase in CD3+CD56+ and a decrease in B lymphocytes. Patients with an increased CD4/CD8 ratio have a better chance of responding to neoadjuvant chemotherapy.

Heme oxygenase-1: role in brain aging and neurodegeneration.

The mechanisms responsible for excessive iron deposition and mitochondrial insufficiency in the aging and degenerating nervous system remain poorly understood. Heme oxygenase-1 (HO-1) is a 32kDa stress protein that degrades heme to biliverdin, free iron and carbon monoxide. Our laboratory has shown that cysteamine, dopamine, beta-amyloid, IL-1beta and TNF-alpha up-regulate HO-1 followed by mitochondrial sequestration of non-transferrin-derived 55Fe in cultured rat astroglia. In these cells and in rat astroglia transfected with the human HO-1 gene, mitochondrial iron trapping is abrogated by the HO-1 inhibitors, tin-mesoporphyrin and dexamethasone. We determined that HO-1 immunoreactivity is enhanced greatly in neurons and astrocytes of the hippocampus and cerebral cortex of Alzheimer subjects and co-localizes to senile plaques and neurofibrillary tangles (NFT). HO-1 staining is also augmented in astrocytes and decorates neuronal Lewy bodies in the Parkinson nigra. Collectively, our findings suggest that HO-1 over-expression contributes to the pathological iron deposition and mitochondrial damage documented in these aging-related neurodegenerative disorders. We recently observed that, paradoxically, HO-1 mRNA levels are markedly suppressed in peripheral lymphocytes of patients with early sporadic Alzheimer disease and may thus provide a useful biological marker of this condition.

Enhanced expression of IFN-gamma mRNA in CD4(+)or CD8(+)tumour-infiltrating lymphocytes compared to peripheral lymphocytes in patients with renal cell cancer.

The mRNA expression of the cytokines IFN-γ, IL-10 and TNF-α and the proapoptotic factor Fas ligand (FasL) was compared in freshly isolated CD4+and CD8+tumour-infiltrating lymphocytes (TIL) and simultaneously obtained autologous CD4+and CD8+peripheral blood lymphocytes (PBL) from 20 patients with renal cell carcinomas (RCC). TIL were isolated from mechanically disaggregated tumour material and PBL from peripheral blood by gradient centrifugation. The cells of the interphase were depleted from tumour cells with anti-human epithelial antigen magnetic beads and then positive selection was performed with anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of cytokine and FasL mRNAs was determined by using a PCR-assisted mRNA amplification assay. In the CD4+TIL from the 20 patients with RCC, levels of mRNAs encoding for IFN-γ (P≤ 0.001), IL-10 (P≤ 0.05), and FasL (P≤ 0.001) were significantly higher than in the autologous CD4+PBL. Comparison of CD8+TIL and CD8+PBL revealed a significant higher expression of IFN-γ (P≤ 0.001), IL-10 (P≤ 0.01) and FasL mRNAs (P≤ 0.001) in the former. However, TNF-α mRNA levels were significantly lower in the CD8+TIL than in the CD8+PBL (P≤ 0.05). These data reflect a general in vivo activation of RCC infiltrating lymphocytes in the tumour surrounding. © 2000 Cancer Research Campaign

Possible association between Taenia solium cysticercosis and cancer: increased frequency of DNA damage in peripheral lymphocytes from neurocysticercosis patients

Helminths, particularly some Schistosoma species, have been associated with cancer in humans. Neurocysticercosis, produced by cysticerci of the helminth Taenia solium, has been associated with the emergence of brain tumours and haematological malignancies. Local tumours, such as glioblastoma, could be explained by the induction of DNA damage in cells surrounding the cysticercus and chronically exposed to an inflammatory host response. However, systemic effects such as haematological malignancies are not easy to understand. The present work was conducted in Mexico to find out whether DNA damage arises in peripheral lymphocytes in patients with neurocysticercosis. We utilized a highly sensitive technique to analyse chromosomal aberrations, in-situ hybridization with probes against chromosomes 1, 2 and 4, and in addition the blocked-cytokinesis technique was used to determine the formation of micronuclei, a peculiar form of DNA damage. The study was made in lymphocytes from 8 patients before and after the administration of praziquantel, 1 of the 2 drugs used for neurocysticercosis treatment. The frequencies of chromosome aberrations and micronuclei in peripheral blood lymphocytes were higher in the infected patients as compared to those observed both in healthy donors and in the group of patients after praziquantel therapy. Our results suggest that chromosome aberrations induced in peripheral cells during neurocysticercosis could be associated with the development of haematological neoplasias.

Aneuploidy index in blood: a potential marker for early onset, androgen response, and metastasis in human prostate cancer

Objectives. To investigate whether the frequency of chromosome abnormalities in peripheral blood lymphocytes defined as the aneuploidy index in blood (AnIB) can be used as a clinical marker of early age onset, androgen response, and metastasis in human prostate cancer. Methods. Peripheral blood samples were collected from 80 patients with prostate cancer, and chromosome preparations were made from 72-hour cultures after mitotic block. The AnIB of 59 informative cases was compared with several parameters, including age at disease onset, Gleason grade of tumor, clinical stage of tumor, metastasis, and prostate-specific antigen (PSA) level. Results. Patients with AnIB levels greater than 3 had a significantly higher incidence of metastasis ( P = 0.022), androgen-independent disease ( P = 0.002), and early age at disease onset (age at diagnosis less than 65 years) ( P = 0.002) compared with the patients with lower AnIB (less than 3) levels. In addition, patients with AnIB levels greater than 5 had higher PSA levels (greater than 20 ng/mL) ( P = 0.029) than patients with AnIB levels less than 5. Conclusions. Chromosome abnormalities can be detected in the peripheral lymphocytes of patients with prostate cancer, and AnIB can be used as an early diagnostic and predictive marker for prostate cancer metastasis and androgen-independent disease.

Flow cytometric analysis of IL-6 receptors on peripheral lymphocytes in patients with primary biliary cirrhosis

Interleukin-6 receptors (IL-6R) and interleukin-1 receptors (IL-1R) on lymphocyte surfaces were analyzed, using flow cytometry and dye-labeled IL-6 and IL-1 beta, to examine the clinical and immunological significance of these receptors. Incubation of peripheral blood mononuclear cells in the presence of mitogen resulted in a remarkable increase of lymphocytes expressing the IL-6 and IL-1 beta receptors on the cell surface. The increase in lymphocytes bearing these cytokine receptors may reflect an increase in stimulated lymphocytes. When peripheral blood from patients with primary biliary cirrhosis (PBC) was examined for these receptors, the percentage of IL-6R positive cells was significantly higher in the patients than in healthy controls (P < 0.01). The increase in IL-6R positive cells was only significant for the T lymphocyte fraction (P < 0.01). No significant change in IL-1R was observed. There was a significant positive correlation between the percentage of IL-6R positive T lymphocytes and the titer of antimitochondrial antibody in patients with PBC. These findings concerning IL-6R may be noteworthy elucidating autoimmune etiological features of PBC.

Flow cytometric analysis of IL‐6 receptors on peripheral lymphocytes in patients with primary biliary cirrhosis

Flow cytometric analysis of IL‐6 receptors on peripheral lymphocytes in patients with primary biliary cirrhosis