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Few data arc available on the blood lymphocyte response to revaccination in man. The anamnestic response to tetanus toxoid challenge was evaluated by a variety of techniques during the first week after revaccination. Out of twenty subjects used, eight were evaluated before and 5 days after the injections (days 1-8), Analytical cell electrophoresis showed important variations in the B and two T lymphocyte populations. The B cell percentages, assessed by EAC-rosettes and electrophitretic mobility, were found to decrease by days 2 and 3, and return to former levels by day 5, when a rise in specific antibodies was detected. A similar response was found in the T1 population, generally considered to be composed of low affinity E-rosette-forming cells. Conversely, a significant increase (50-100%) in circulating T2 lymphocytes (active rosette-forming cells) was found by days 2 and 3, followed by a rapid decrease of these 'differenciated' cells. The increase in the T2 lymphocytes appeared earlier in skin test positive subjects. These changes were correlated with E-rosettes, mitogen stimulation, peripheral leucocyte migration inhibition and transformation in the presence of the antigen. EA-IgG rosettes and ADCC varied similarly. These results may indicate a significant non-specific cell mobilization following revaccination. [ABSTRACT FROM AUTHOR]

HLA-D/DR (Ia) glycoproteins were identified in human breast carcinoma and normal mammary gland cells by means of an anti-Ia monoclonal antibody. Two techniques were used: (1) immunoperoxidase staining performed on histological sections and (2) Ia glycoproteins were isolated as follows: firstly by radioactive labelling of isolated cells, then by filtratien on Sephadex G25, followed by Lens culinaris chromatography, and immune complex formation and then elution on protein A-Sepharose. Lastly, the immune complex was studied by chromatofocusing. Both techniques revealed that Ia expression was found in carcinoma cells, but not in normal cells. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is a severe neuromuscular disease causing a progressive muscle wasting due to mutations in the DMD gene that lead to the absence of dystrophin protein. Adeno-associated virus (AAV)-based therapies aiming to restore dystrophin in muscles, by either exon skipping or microdystrophin expression, are very promising. However, the absence of dystrophin induces cellular perturbations that hinder AAV therapy efficiency. We focused here on the impact of the necrosis-regeneration process leading to nuclear centralization in myofiber, a common feature of human myopathies, on AAV transduction efficiency. We generated centronucleated myofibers by cardiotoxin injection in wild-type muscles prior to AAV injection. Intramuscular injections of AAV1 vectors show that transgene expression was drastically reduced in regenerated muscles, even when the AAV injection occurred 10 months post-regeneration. We show also that AAV genomes were not lost from cardiotoxin regenerated muscle and were properly localised in the myofiber nuclei but were less transcribed leading to muscle transduction defect. A similar defect was observed in muscles of the DMD mouse model mdx. Therefore, the regeneration process per se could participate to the AAV-mediated transduction defect observed in dystrophic muscles which may limit AAV-based therapies., (© 2022. The Author(s).)

Adeno-associated virus (AAV) transduction efficiency depends on the way in which cellular proteins process viral genomes in the nucleus. In this study, we have investigated the binding of nuclear proteins to the double stranded D (dsD) sequence of the AAV inverted terminal repeat (ITRs) by electromobility shift assay. We present here several lines of evidence that transcription factors belonging to the RFX protein family bind specifically and selectively to AAV2 and AAV1 dsD sequences. Using supershift experiments, we characterize complexes containing RFX1 homodimers and RFX1/RFX3 heterodimers. Following transduction of HEK-293 cells, the AAV genome can be pulled-down by RFX1 and RFX3 antibodies. Moreover, our data suggest that RFX proteins which interact with transcriptional enhancers of several mammalian DNA viruses, can act as regulators of AAV mediated transgene expression.

Objectives: The French Society of Otorhinolaryngology (SFORL) set up a work group to draw up guidelines for initial staging of head and neck squamous cell carcinoma. Locoregional and remote extension assessment are dealt with in two separate reports. The present part 3 deals with the assessment of frequent associated symptoms and pathologies, requiring early treatment and the collection of data on a certain number of clinical and paraclinical parameters for therapeutic decision-making in the multidisciplinary team meeting., Materials and Methods: A multidisciplinary critical analysis of the literature was conducted. General assessment here covers screening, assessment and initial management of the following: usual risk factors (smoking, alcohol, HPV), the most frequent medical comorbidities, nutritional status, social and psychological status, dental status, pain and possible anemia. As oncologic management frequently associates surgery, radiation therapy and chemotherapy, the underlying examinations should be early, as part of initial staging. The levels of evidence for the examinations were estimated so as to grade guidelines, failing which expert consensuses were established., Results: The high rates of pain, malnutrition and anemia call for systematic screening and early management, especially as rapidly effective treatments exist. Assessing comorbidity and social and psychological status enables general health status to be assessed, along with possible contraindications to the usual treatments. Tracheal intubation problems may require intubation under flexible endoscopy or jet-ventilation by inter-cricothyroid catheterization from the diagnostic endoscopy stage. Assessment and adapted dental care should be conducted if radiation therapy is likely or certain., Conclusion: Early management of symptoms and comorbidity and anticipation of subsequent treatment are intended to shorten initial staging time and to collate the data needed for therapeutic decision-making. This assessment should be performed at the same time as the locoregional and remote extension assessment, and is obviously to be adapted according to tumoral extension stage and the possible treatment options., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Merkel cell carcinoma (MMC) is a rare primary neuroendocrine skin tumor associated with a poor prognosis. MMC is histologically similar to small cell lung carcinoma. The incidence of MCC is increasing with a median age of discovery of approximately 70 years old. Treatment of MCC, which usually occurs in sun-exposed skin, includes surgery with reconstruction associated with radiotherapy and/or chemotherapy for advanced stages including widespread metastases. We report two cases of Merkel cell carcinoma in 76 and 84 year old patients and describe the natural history of this cancer and management in geriatric patients. Merkel cell carcinoma (MCC) is a rare cancer with high malignant potential that mainly affects people over the age of 65. It must be diagnosed early to improve the prognosis. The survival rate at 5 years for local or regional invasion is 65%. The median survival for metastasized stages is 10 months. The therapeutic regimen recommended for the treatment of MCC is influenced by the geriatric status of patients, and a multidisciplinary oncogeriatric approach could be of interest.

Background Aims: The clinical benefits of extracorporeal photochemotherapy (ECP) are well recognized, but its clinical use is limited by logistical difficulties, especially because of the need to perform repeated aphereses. The cryopreservation of mononuclear cells could allow maintenance of the ECP schedule while reducing the number of aphereses. The aim of this work was to assess whether previous cryopreservation impairs the immunomodulatory function of ECP-treated peripheral blood mononuclear cells (PBMC)., Methods: Fresh or previously cryopreserved PBMC were exposed to ECP and added on day 0 into a mixed leukocyte reaction. Proliferation of alloreactive lymphocytes was measured by carboxyfluorescein succinimidyl ester (CFSE) dye dilution. Apoptosis was quantified by annexin-7AAD staining., Results: ECP-induced apoptosis was slightly increased in cryopreserved cells but the kinetics of apoptosis were similar to fresh cells. Lymphocytes stimulated in the presence of ECP-treated PBMC displayed a significant decrease in proliferation. The suppression was enforced when ECP-treated cells had been activated previously by allogeneic stimulation. Cryopreservation before ECP exposure did not impact apoptosis triggering or anti-proliferative properties of ECP-treated cells., Conclusions: Cryopreservation before ECP does not impair the immunomodulatory effects of treated cells. These data warrant investigation of the clinical use of cryopreserved PBMC for ECP.

Radiation therapy plays a central role in the treatment of glioblastoma, but it is not curative due to the high tumor radioresistance. Phosphatidyl-inositol 3-kinase/protein kinase B (Akt) and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathways serve to block the apoptosis process, keeping cells alive in very toxic environments such as chemotherapy or ionizing radiation. In the present study, from a panel of 8 human malignant glioma cell lines, investigations on the relationship between intrinsic radioresistance and Akt or STAT3 basal activation were done. Secondly, the impact of down-modulation of Akt or STAT3 signaling on in vitro intrinsic radiosensitivity was evaluated. Using a clonogenic cell survival assay, our results revealed a significant correlation between the basal Akt activation and the surviving fraction at 2 Gy (SF2). In contrast, no correlation was found between STAT3 activation and SF2. According to this, down-modulation of Akt with a specific chemical inhibitor (Akt inhibitor IV) demonstrated a significant enhancement of radiation sensitivity on glioma cells in a clonogenic survival assay. On the contrary, down-modulation of STAT3 signaling with a specific chemical inhibitor (JSI-124) or a neutralizing gp130 antibody failed to radiosensitize glioma cells. These data indicate that the Akt intercept node could be a more relevant therapeutic target than STAT3 for radiosensitizing human malignant glioma.

For most therapeutic strategies using MSC, the preliminary amplification is carried out in media containing fetal calf serum (FCS). The theoretical health risk of using a xenogenic serum, a recent practice for which we have limited data, cannot be underestimated, while amplification using human serum (HS) remains controversial. At present, the available information on multipotentiality, self-renewal, and transplantability does not permit the selection of FCS rather than HS. Cellular modifications observed during cell passage seem to indicate a gradual impairment of cells in relation to native MSC, suggesting the making of short cell cultures without necessarily trying to reinfuse a high number of MSC in patients. With this approach, the volume of HS required would remain limited. While clinical studies have already started, many problems remain, such as evaluating the quality of the initial mesenchymal compartment and the biological properties of the cell suspension with FCS compared to those with HS, and depending on culture time.

Objective: The progress made in the supportive care of allografts and the identification of mesenchymal stem cells in adult human bone marrow (BM) has prompted renewed interest in the use of BM as a form of cell therapy. With the aim of optimizing the collection of BM cells, we evaluated the hematopoietic and mesenchymal immature cell contents of BM hematon units (HUs), which usually are eliminated during graft processing., Materials and Methods: Hematopoietic CD34+ progenitors from HU and buffy coat (BC) compartments were characterized in short-term culture. The sorted CD34+CDw90(Thy-1)+ primitive subset was assessed in colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, then further characterized by the expression of additional antigens. In parallel, we evaluated the colony-forming unit fibroblast (CFU-F) number and phenotyped the fresh adherent (D1-3) cells., Results: The plating efficiencies of CD34+ cells derived from HU and BC were identical. However, the HU CD34+CDw90(Thy-1)+ subset was enriched in colony-forming unit megakaryocyte (2.3x), LTC-IC (4.6x), and cells coexpressing CD105 (5x). We found a higher frequency of CFU-F (4.7x), considered to be the mesenchymal stem cell-containing population, correlated with an enrichment in fresh adherent (CD45/GPA)-CD14- cells., Conclusions: We show for the first time that functional properties of the CD34+CDw90+ subset are related to its in vivo location in HU, which may represent the BM mesenchymal reserve compartment. The location in HU of 35.6%, 59.1%, and 58.7% of CD34+ cells, CD34+CDw90+ LTC-IC, and CFU-F, respectively, justifies the development of a procedure to collect them in order to reduce the therapeutic BM volume.

Background: Undernutrition is a main cause of immunodeficiency. Many confounding factors limit the interpretation of immune function in hospitalized elderly patients., Objective: We compared the effects of short-term fasting and refeeding on lymphocyte subset distribution and neutrophil function in healthy subjects., Design: Seven young adult (x +/- SE age: 24 +/- 2 y) and 8 elderly (71 +/- 3 y) subjects were fed standardized diets (1.6 x predicted resting energy expenditure; 16% protein) for 7 d. They then fasted for 36 h and were refed for 4 h (42 kJ/kg). Lymphocyte subsets were quantified by using fluorochrome-conjugated monoclonal antibodies. Neutrophil chemotactic migration was evaluated by using a 2-compartment chamber. Neutrophil reactive oxygen species production was measured by using a luminol-amplified chemiluminescence assay and oxidation of 2'7'-dichlorofluorescein diacetate., Results: Baseline total and cytotoxic T lymphocyte subpopulations were lower in elderly than in adult subjects (P < 0.01). Nutritional state had a significant effect (P < 0.05) on total, helper, and cytotoxic T and B lymphocyte counts in all subjects, and the response of lymphocyte subpopulations to nutritional fluctuations was significantly affected by age. The chemotactic index was lowered by fasting in both groups (P < 0.05 compared with basal values). After refeeding, neutrophil migration was restored in adult but not elderly subjects. The superoxide anion production rate increased with fasting and reverted to prefasting values with refeeding in both groups (P < 0.05). Fasting induced a significant decrease in hydrogen peroxide production in stimulated neutrophils that was reversed by refeeding in adult but not elderly subjects., Conclusion: The lack of response of lymphocyte subpopulation counts and neutrophil function to nutritional changes may help to explain the proneness of elderly persons to infection.

Background: Previous reports suggest that correcting the malnourished state is more difficult in elderly people than in younger ones and that protein requirements may be higher in elderly than in younger adults., Objective: The aim of this study was to establish whether malnourished old rats respond to protein-supplemented nutritional repletion as do young adult rats., Design: Adult (3 mo old) and old (22 mo old) rats were submitted to dietary restriction programs that induced similar metabolic and nutritional alterations. Malnourished adult and old rats were then killed (R groups) or refed for 1 wk with a high-protein diet (HPD; 23% protein) or a very-high-protein diet (VHPD; 27% protein). Control groups at both ages were fed ad libitum throughout the experiment. Effects of food repletion were evaluated in terms of protein metabolism, intestinal histomorphometry, and nonspecific immune status., Results: In adult rats, HPD sufficed to increase body weight and restore basal values of liver weight and protein content (P: < 0.01 compared with the R adult group), nitrogen balance (P: < 0.01 compared with the R adult group), and hydrogen peroxide production by polymorphonuclear neutrophils and monocytes (P: < 0.01 compared with the R group); VHPD had no supplementary effect except on nitrogen balance. In old rats, HPD was less effective and greater benefit was observed with VHPD in terms of body weight gain (10%; P: < 0.01 compared with the old group fed HPD), albuminemia, muscle weight and protein content, plasma arginine concentration, and hydrogen peroxide production by stimulated polymorphonuclear neutrophils and monocytes compared with the old R group (P: < 0.01)., Conclusion: Aging is a significant variable affecting the response to nutritional support.

The antigen receptor genes studied (immunoglobulin gene for B-cells, and T-cell receptor -beta or -gamma gene for T-cells) represent the most powerful tools for diagnosing the clonality of a lymphoid lineage. We have clonotyped 23 cutaneous T-cell lymphomas and 5 were found to be clonotypically all heterogeneous. Analysis of each patient was performed either from serial skin biopsies taken several months apart or from different tumor samples. In these cases, T-cell lymphoma clonotypic heterogeneity was demonstrated and was especially evident when examining different tumor sites. Moreover, in one case, a biogenotypic population (immunoglobulin and T-cell receptor-rearranged) was found. This unexpected high frequency of T-cell clonal heterogeneity (22%) could be explained either by the evolution of subclones from a single undifferentiated malignant cell or by the independent transformation to cancer of 2 or more lymphocytes, though the latter seems less likely. Clonotypic heterogeneity seems to be as frequent in T-cell lymphomas with cutaneous lesions as in B-cell leukemias.

Cyclosporine A (an immunosuppressive agent) decreases Ia lymphoid differentiation marker in female Sprague-Dawley rats with N-nitroso-N-methylurea-induced mammary tumors. Presence of lymphoid differentiation antigens was determined on mammary tumor cells and lymphoid cells from bone marrow, spleen, and peripheral blood by flow cytometric analysis. Quantification of Ia antigen expression was also performed by affinity chromatography and chromatofocusing in mammary tumors. A significant decrease in Ia antigen expression by mammary tumors of animals treated with cyclosporine A was noted with the two different methods. Cyclosporine A acts as an antagonist to prolactin receptors in such hormone-dependent mammary cancer. Our results should prove very useful in understanding the mechanisms of prolactin regulation of Ia antigen in tumorigenesis of the mammary gland.

The present work compared the blood variations in some committed stem cells (CSC), and corresponding differentiated WBC during the first three courses of a 6-day sequential chemotherapy given to 16 breast cancer patients for 28 days each. The granulomonocytic and lymphocytic colony-forming unit levels in blood were significantly lowered in cancer patients before treatment. These CSC appeared to have cyclic variations following each course of chemotherapy. In granulomonocytic colony-forming units, a nadir was observed by Day 15, followed by a sharp rebound above the initial values by Days 22 to 24, which was not affected in magnitude by the continuation of treatment. In lymphocytic colony-forming units, the Day 1 level increased with the continuation of chemotherapy. The initial decrease was less marked, but by Day 15 a minimal level was also observed, followed by a progressive increase to reach a maximum by Day 28. Leukocytes and granulocytes reached a nadir by Days 16 to 17 and recovered by Day 25. The cyclic evolution of monocytes was less apparent as was that of lymphocytes; however, there was less restoration of monocytes and lymphocytes than of granulocytes at the end of the resting period. This study showed: (a) an apparent relationship between the level of CSC in blood, and subsequent variations in the corresponding mature cells of the same lineage; and (b) a weak interval of time between the nadir and peaks of CSC and the corresponding mature cells, which was more evident in the granulocytic lineage. These observations seemed to be of physiological importance, but the possible prediction value of peripheral granulomonocytic colony-forming unit peak magnitude following treatment remains to be established.

Class II HLA antigen expression in breast carcinoma and normal breast gland cells was compared using a method more accurate than immunofluorescence. This new method involves labeling membrane proteins with 131I and the anti-Class II HLA monoclonal antibody with 125I. The isolation and purification of the doubly labeled (125I-131I) immune complex was performed by affinity chromatography and chromatofocusing successively. When the specific activity of glycoproteins is known, the amount of glycoproteins which bind specifically to the anti-Class II HLA monoclonal antibody can be deduced. In breast carcinoma cells, 1.5 to 2% of the purified glycoproteins bind specifically to the monoclonal antibody, whereas less than 0.3% of normal breast gland cells binds. In contrast, leukemic cells, of which 80 to 90% possess Class II HLA antigens, 2 to 3% of Class II HLA glycoproteins bind specifically with the anti-Class II HLA monoclonal antibody.

The authors describe a child who presented multiple congenital malformations affecting the cranio-facial region and the extremities associated with a rare skin lesion (incontinentia pigmenti achromians).

A 50-year-old man was admitted to the hospital with a diagnosis of eczema. Using study of T-cell receptor gene rearrangement on skin biopsies, a diagnosis of mycosis fungoides was made, then confirmed by evolution. This observation provides the opportunity to discuss the usefulness of molecular biology for the early diagnosis of mycosis fungoides.

beta-Thromboglobulin (beta TG) plasma levels were determined in 52 female breast cancer patients at different stages and in 39 healthy controls (22 women and 17 men) of similar age distribution. Beta TG levels were high (mean +/- SD:61.6 +/- 59.1 ng/ml) in patients before any treatment compared to controls (mean +/- SD:21.2 +/- 7.4 ng/ml) and the difference was statistically significant (p less than 0.001). No correlation with disease stage was observed. No other coagulation parameters were abnormal except fibrinogen, which increased. Fibrinogen degradation products (FDP) also increased but only in metastatic patients. Chemotherapy appeared to induce a considerable decrease in initial values at the end of the first cycle without modifying the platelet count. In addition, an attempt was made to correlate the beta TG plasma level investigated serially for several months with disease evolution.