CD4 lymphocyte count and CD4/CD8 ratio in elderly long-term care patients with oropharyngeal dysphagia: comparison between oral and tube enteral feeding.

Many institutionalized elderly patients are at risk of undernutrition as a result of oropharyngeal dysphagia (OD) that possibly affects their immunological status. Tube enteral fed (TEF) patients on controlled intake of nutrients enables us to evaluate the effect of inadequate nutrition on the immune system in the orally fed elderly with OD. The aim of our study was to compare CD4 lymphocyte count and CD4/CD8 ratio between these two differently fed groups. Twenty-eight orally fed patients with OD in the Functional Outcome Swallowing Scale (FOSS) stage 2 (group A) and 17 TEF subjects (group B) were studied. CD4 and CD8 counts were determined by flow cytometry. Nutritional markers (albumin, hemoglobin, and basal metabolic index) were recorded for each group. The Charlson index was used for comparison of comorbidity between the two patient groups. The average count of CD4 lymphocytes was significantly lower in group A than in group B (754 +/- 365 vs. 1032 +/- 404 cells/ml, p < 0.01). Six patients in group A (21%) had a CD4 count of less than 400 cells/ml (lower threshold) while all the patients in group B had a CD4 count of over 500 cells/ml (p < 0.001). The CD4/CD8 average ratio was also significantly lower in group A (p < 0.008). Nutritional markers were within normal limits with no difference between the groups. These results confirm our presumption that a low CD4 lymphocyte count and a low CD4/CD8 ratio could prevail among elderly frail patients with dysphagia. This supports the view that under an apparently satisfactory nutritional profile these patients may be in a state of undernutrition that negatively influences their immunodefense.

Altered subcellular localization of p53 in estrogen-dependent and estrogen-independent breast cancer cells.

LCC2, an estradiol-independent tamoxifen (Tax)-resistant subline of MCF-7 human breast cancer cell line, is resistant relatively towards Tax and methotrexate (Mtx). The purpose of the present study is to evaluate the role of p53 in determining this resistance. While MCF-7 is sensitive to and undergoes apoptosis, as determined by propidium iodide stain, by Tax and Mtx, LCC2 is resistant to apoptosis induction by these agents. Both cell lines undergo apoptosis and are sensitive equally to doxorubicin (Adr). p53 cDNA of both sublines was evaluated by polymerase chain reaction (PCR) amplification and sequencing and was found to be of wild-type. p53 mRNA, as well as protein, are elevated markedly in LCC2 as compared to MCF-7 cells. p53 expression was increased by estradiol and Adr, not changed by Mtx, and decreased by Tax and estradiol-deprivation in both sublines. p53 modulation by the various agents, in both sublines, was evaluated by cytochemical staining and subcellular fractionation. This analysis showed that p53 is localized mainly in the nuclear fraction in MCF-7 cells, and in the cytoplasmatic fraction in LCC2 cells. Doxorubicin induced apoptosis in MCF-7 cells along with increase in its nuclear fraction. In contrast, LCC2 underwent apoptosis by Adr despite its cytoplasmatic sequestration. These experiments demonstrate that p53 is sequestered to cytoplasm in the estrogen-independent, Tax-resistant LCC2 cells. However, the differences in apoptotic rate between MCF-7 and LCC2 cells do not seem to be dependent on p53. The LCC2 cell line may serve as a useful model for the study of the mechanism of cytoplasmatic sequestration of wild type (wt) p53, its physiologic consequences, and its relation to estrogen-independence or Tax resistance of breast cancer cells.

Expression of wild-type p53 and Bcl-2 family genes oscillates with recurrent remission and relapse in an unusual case of low-grade lymphoma.

Downregulation of apoptosis has been proposed as a mechanism of clonal expansion in low-grade B cell neoplasms. We have previously described an unusual case of CD5+ B cell lymphoma characterized by cycles of leukemic phase alternating with spontaneous remission. In the present study, we examined the involvement of apoptosis-related proteins in the progression of this cyclic lymphoma ex vivo. During the leukemic phases, the clonal cells were activated blasts expressing elevated levels of wild-type (wt) p53, Bcl-2, Bcl-x(L), and Bax, while Bak expression increased during the decline of lymphocytosis. Bax heterodimerized with Bcl-2 but not with Bcl-x(L). The anti-apoptotic Bcl-2/Bax heterodimers peaked during early leukemic phases and declined during regression. The elevation in Bcl-2, Bcl-x(L) and Bax expression during early leukemic phases seems to result from cell activation since a similar increase was induced by activating the remission phase leukemic cells in culture. The data suggest that wt p53, Bcl-x(L), and Bcl-2/Bax heterodimers support the accumulation of activated leukemic cells during the leukemic phases, while Bax and Bak may be involved in their decline during regression.

Placental apoptosis in normal and abnormal pregnancies.

The aim of this study was to demonstrate apoptosis in the human placenta in normal and abnormal pregnancies. The percentage of apoptotic cells in placental samples was quantified by analyzing the cell cycle of nuclei stained with propidium iodide using a flow cytometer. No significant difference in the percentage of apoptotic cells was observed comparing the group of normal pregnancies (first and second trimesters) with those of missed abortions. There was also no difference in the incidence of apoptosis comparing placental samples obtained from chromosomally normal and abnormal pregnancies. Yet, there was a significant increase in the incidence of apoptosis in placental samples obtained from second and third trimesters as compared with those obtained from the first trimester (p<0.04 and p<0.01, respectively). There was also a significant increase in the incidence of placental apoptosis in the third as compared with the second trimester (p<0.03).

Expression of the p53 gene and apoptosis in gestational trophoblastic disease.

In order to understand the involvement of the p53 tumour suppressor gene in the pathogenesis of gestational trophoblastic disease (GTD), we investigated its genetic status, protein expression and its role in apoptosis in samples of complete and partial hydatidiform mole as compared with those of normal placenta. Direct sequencing of polymerase chain reaction (PCR) products of the coding and non-coding regions of the p53 gene demonstrated no mutations in any of the studied samples. Immunohistochemical studies revealed increased expression of the p53 protein predominantly in the nuclei of villous cytotrophoblasts. This over-expression of p53 was found in all samples of complete mole, in 50 per cent of partial mole samples and in about 30 per cent of normal placenta cases, although no significant difference in the staining intensity and pattern was observed. An in situ detection of DNA nicking (TUNEL) staining, demonstrating apoptosis, was also detected predominantly in villous cytotrophoblasts and in stromal areas. The per centage of apoptotic cells in all studied samples, determined by flow cytometry, demonstrated a significant increase in apoptotic cells in samples of complete and partial hydatidiform mole compared with those of normal placenta (P< 0.0003 and P< 0.004, respectively). In conclusion, the current study may provide a possible explanation to the pathogenesis of GTD, probably associated with extensive p53-dependent apoptosis to modulate excessive trophoblastic proliferation.

Polymorphisms of the p53 gene in women with ovarian or endometrial carcinoma.

The p53 gene is frequently mutated in various human tumors. Polymorphism is an additional genetic alteration observed in exons and introns of the p53 gene of normal tissues and tumors. Distributions of alleles of three common polymorphisms of the p53 gene; a 16 bp duplication in intron 3, codon 72 of exon 4 and a sequence in intron 6, were studied in peripheral white blood cells (WBC) of patients with ovarian or endometrial carcinomas. The analysis was performed by PCR and direct sequencing. The 100% linkage observed between the most common haplotypes of each polymorphism in healthy subjects was lower in the patients. A significant difference was observed between frequencies of genotype and haplotype combinations in patients with ovarian carcinoma and endometrial carcinoma. The incidence of heterozygosity was increased in ovarian carcinoma and decreased in endometrial carcinoma. Our results suggest that the p53 gene may be involved in susceptibility and predisposition to various cancers not only by mutations but also by preferential presentation of polymorphic alleles.

P53 gene mutation in a T-acute lymphoblastic leukemia cell line (loucy) with t(16:20) and 5q- chromosomal aberrations.

A human T-acute lymphoblastic leukemia (ALL) cell line (Loucy), derived from cells from a patient with resistant ALL with a t(16:20) and 5q- chromosomal aberrations was evaluated for p53 gene alterations and expression. Western blot analysis of p53 showed elevated levels of the protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and direct sequencing identified a point mutation at codon 272 (GTG --> ATG) of the p53 gene. Possible molecular mechanisms underlying these alterations and their role in the establishment of this cell line and in leukemogenesis in general are discussed.

Molecular alterations in the TP53 gene of peripheral blood cells of patients with chronic myeloid leukemia.

The TP53 gene has been extensively studied in patients with chronic myeloid leukemia (CML), both in chronic phase and in blast crisis. Mutations in the gene were found in up to 30% of the patients, especially among those in blast crisis. We report the results of an analysis of 29 blood samples from CML patients: 8 samples from chronic phase patients, 8 from patients in the accelerated phase, and 13 from patients in blast crisis. By using genomic DNA, we sequenced PCR products of the coding exons and most introns of the TP53 gene, finding genetic changes in 30% of the blast crisis samples and 12% in chronic phase. All mutations were found in introns and were previously unreported. Immunocytochemical studies revealed accumulation of TP53 in blood cells of samples both from chronic phase and blast crisis patients. Since these samples had no TP53 mutations, we believe that wild type TP53 accumulates in blood cells of CML patients. Our results, therefore, indicate that molecular changes in coding regions of the TP53 gene are rare. The significance of the abundance of intronic changes should be investigated further. Accumulation of wild type TP53 in CML cells may indicate an additional mechanism involving this gene in the pathogenesis of this disease.

Clinical implications of p53: effect on prognosis, tumor progression and chemotherapy response.

The p53 tumor suppressor gene is frequently mutated in most human malignancies. These mutations have been associated with clinical outcome for various cancer types. Since this gene's main function is to guard the integrity of the genome, its clinical relevance, i.e. its role in carcinogenesis and chemotherapy-drug resistance, have been extensively studied. These data and the p53 protein as a potential target for new treatment strategies, are reviewed based on acquired knowledge of the structure function of the p53 protein.

Immunosuppressive effect of uterine fluid on spermatozoa-stimulated mixed lymphocyte cultures.

Spermatozoa, obtained from 20 healthy individuals, were incubated with crude uterine fluid or with a purified IgG fraction obtained from uterine fluid before adding them to cultures with allogeneic lymphocytes. Untreated spermatozoa induced a proliferative 1.5- to 6-fold increase, while the spermatozoa treated with crude uterine fluid or with purified IgG fraction caused 84-100% suppression of lymphocyte blastogenesis. These results suggest the existence of an endogenous uterine fluid factor, apparently IgG, which causes inhibition of lymphocyte proliferation induced by spermatozoa in the uterine lumen.

A novel polymorphism in intron 6 of the human p53 gene: a possible association with cancer predisposition and susceptibility.

We present a novel polymorphic 8-bp sequence in intron 6 of the p53 gene that maps between bp 55 and 62 of the 3' end of exon 6. Of normal blood samples, 32% were heterozygotic for this polymorphism and display a NN' genotype, whereas 68% of the population is homozygotic for the N genotype. The rare homozygotic genotype N' was detected only in four blood samples of cancer patients. Peripheral blood of gastrointestinal (GI) and breast tumor patients demonstrated a higher incidence of heterozygosity (50%) than that of normal individuals. Analysis of the distribution of this polymorphism in tumor samples showed loss of heterozygosity (LOH). This LOH during tumor progression could exhibit preference to each one of the polymorphic alleles. The rare presentation of one allele and the increased incidence of heterozygosity in carcinoma patients may suggest an association between this polymorphism with cancer predisposition and susceptibility. The fact that genetic alterations occurring in noncoding regions may play a role in tumor development only further increases the extent of involvement of p53 in carcinogenesis.

p53 mutations in matched primary and metastatic human tumors.

Mutations in the p53 tumor suppressor gene have been found to be the most frequent genetic alterations in human malignancies. To further examine the idea that neoplastic progression is associated with mutations in the p53 gene, we analyzed matched primary and metastatic tumor samples. The samples included 15 pairs of breast cancer and metastases to lymph nodes, four pairs of gastrointestinal adenocarcinomas and metastases to liver, one colon adenocarcinoma and metastasis to a lymph node, and one lung carcinoma and metastasis in the pleura. Genomic DNA or cDNA from each tumor sample was amplified by the polymerase chain reaction and labeled by using one biotinylated primer. The DNA strands were separated with magnetic streptavidin beads and sequenced directly. p53 mutations were detected in 11 of 21 patients (52%) in either primary tumors, metastases, or both. In six of these patients the primary tumor and matched metastasis shared the same single mutation. In the other patients an additional mutation in the primary tumor only or a mutation in the metastasis only was observed. Our data suggest that tumor development and progression toward metastasis involves structural alterations in the p53 gene that occur early in carcinogenesis. In some cases, genetic changes in metastatic spreading may also include the appearance of a mutation in a metastasis derived from a primary tumor expressing wild-type p53, a selection of metastatic cells with a single mutation from a primary tumor expressing two different mutations, or loss of heterozygosity.

Immunological profile changes following perioperative autologous vs. homologous blood transfusion in oncologic patients.

To determine the effect of perioperative blood transfusion on immunological parameters, T cells, T-cell subsets, and concanavalin A-induced suppression were measured in 25 patients with colorectal and breast cancer. During the operation, 15 patients received autologous blood and 10 patients had homologous transfusion. The immunological status was again determined after curative surgery. Before surgery, normal percentage of T lymphocytes, decreased ratios of helper/suppressor cells, and impaired con A-induced suppression were found. Following the operation, the helper and suppressor cell percentages reversed to normal, whereas the con A-induced suppression remained impaired. This change was significantly more pronounced in patients who received autologous blood transfusion than in the other group. Autotransfusion has an impact on immune parameters that might prove less detrimental to the clinical outcome in oncologic surgery than homologous transfusion.

Decreased CD45RA T cells in B-cell chronic lymphatic leukemia patients: correlation with disease stage.

T-cell subsets CD4, CD8 and suppressor-inducers (CD45RA) were determined in 20 patients with B-cell chronic lymphatic leukemia (B-CLL). The proportion of CD4 and CD45RA was decreased when compared with T cells from normal subjects. CD8 was markedly increased. The activity of concanavalin A-induced suppressor cells was not significantly different from that of normal controls and was negatively correlated to the percentage of CD4 of B-CLL patients. The selective loss of CD45RA cells was more prominent in patients in advanced Rai stages of the disease (III to IV) than in early stages (0 to II). Six patients of the advanced stages group suffered from autoimmune hemolytic anemia, whereas no patient in the early stages of disease showed an autoimmune phenomenon. Our results may indicate a mechanism of autoimmunity in B-CLL similar to that of patients with autoimmune diseases.

Immunophenotypes and Gene Rearrangements of the T-Cell Receptor in Two Patients with a Large Granular Lymphocyte Disorder.

Large granular lymphocyte disorder (LGLD) is a lymphoproliferative disease, characterized by moderate lymphocytosis with an excess of large granular lymphocytes, neutropenia, anemia, and a variable, but mostly chronic clinical course. We describe two patients with LGLD. One patient presented with symptomatic corticoster-oid-responsive hemolytic anemia, while the other had a chronic course not requiring therapy. The majority of lymphocytes from both patients were CD8 + T lymphocytes. However, the cells from the two patients differed in the molecular pattern of the T cell receptor (TCR), and this may explain the difference in their clinical course.

Preincubation with intravenous lipid emulsion reduces chemotactic motility of neutrophils in cord blood.

Neutrophils from cord blood of healthy term infants were isolated and incubated for 30 min with varying concentrations of intravenous lipid emulsion (ILE) solution (4, 8, 20 mg/ml). In vitro assay of chemotaxis was performed after incubation for 120 min with endotoxin-activated serum (EAS). Neutrophil random motility was unchanged after ILE incubation yet chemotactic factor (EAS)-stimulated motility was significantly reduced in a dose-related pattern.

Tuftsin induces tissue factor-like activity in human mononuclear cells and in monocytic cell lines.

Normal human monocytes and macrophages generate potent procoagulant activity (PCA) resembling tissue factor (TF) in response to various stimuli. In this study we show that tuftsin, a natural stimulator of many functions of monocytes and macrophages, also stimulates a potent PCA in mixed mononuclear cells and monocytes, and a mild PCA in lymphocytes and cell lines of monocytic origin (U937 and THP). No activity was generated by several lymphoid cell lines and HL-60 cells. The PCA resembled TF in that it accelerated clotting through the extrinsic coagulation pathway and was inhibited by concanavalin-A and by monoclonal anti-TF antibodies. The induction of TF-like activity by tuftsin was dose- and time-dependent. It was located in the cell membrane and did not require T cells for expression. Generation of TF-like activity was prevented by actinomycin D, while cytarabine had no effect on this process, suggesting that expression of the activity depends on protein synthesis. Studies with various tuftsin analogs suggest that tuftsin stimulates generation of TF-like activity, as well as other functions of monocytes via the same receptors. The results with the monocytic cell lines show that tuftsin affects mainly mature cells. The induction of TF-like activity in mononuclear cells by tuftsin constitutes an important link between mononuclear cells and the immune and coagulation systems. It may play a major role in the pathogenesis of thromboembolism and fibrin deposition in various inflammatory and immunologic disorders.

Rearrangements in the p53 gene in Philadelphia chromosome positive chronic myelogenous leukemia.

Molecular structural analysis of the p53 gene in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) indicates a significant incidence of gene rearrangements in patients at either accelerated phase or blastic crisis. Southern blot analysis of genomic DNA hybridizing with either genomic or cDNA p53 specific probes indicated that 30% of the CML patients at blastic crisis phase exhibited rearrangements, mostly mapping downstream to the first non-coding exon. This is compatible with the observation that the progression of CML from the chronic to the acute phase involves frequent aberrations in chromosome 17, to which the p53 oncogene has been mapped. Therefore, we suggest that one of the pathways of development of CML to the acute phase is associated with aberrations in the p53 nuclear oncogene.

Common variable immunodeficiency: a family study and therapeutic trial with cimetidine.

Various immunologic parameters were tested in a family of two patients with common variable immunodeficiency. Both patients had low serum immunoglobulin levels, low peripheral B cell and T4 subclass of lymphocytes, and reversed T4/T8 ratio. One of the patients had excessive suppressor activity. Two of the asymptomatic members of the family (the father and one brother) had also low T4/T8 ratio that was not associated with excessive suppressor activity. No linkage between the disease inheritance to HLA could be observed. A study of the T cell helper activity to an antigen, the response to which is regulated by an HLA-linked gene, suggested a defect in the immune response of the two patients and their asymptomatic brother with immunologic disorders. Treatment with cimetidine of the patient with excessive suppressor activity led to an improvement in his clinical state, reduction in suppressor activity, temporary effect on his proliferative response capacity to mitogens, and an increase in the antigen-specific helper activity.