17-AAG and Apoptosis, Autophagy, and Mitophagy in Canine Osteosarcoma Cell Lines.

Canine osteosarcoma is highly resistant to current chemotherapy; thus, clarifying the mechanisms of tumor cell resistance to treatments is an urgent need. We tested the geldanamycin derivative 17-AAG (17-allylamino-17-demethoxygeldanamycin) prototype of Hsp90 (heat shock protein 90) inhibitors in 2 canine osteosarcoma cell lines, D22 and D17, derived from primary and metastatic tumors, respectively. With the aim to understand the interplay between cell death, autophagy, and mitophagy, in light of the dual effect of autophagy in regulating cancer cell viability and death, D22 and D17 cells were treated with different concentrations of 17-AAG (0.5 µM, 1 µM) for 24 and 48 hours. 17-AAG-induced apoptosis, necrosis, autophagy, and mitophagy were assessed by transmission electron microscopy, flow cytometry, and immunofluorescence. A simultaneous increase in apoptosis, autophagy, and mitophagy was observed only in the D22 cell line, while D17 cells showed low levels of apoptotic cell death. These results reveal differential cell response to drug-induced stress depending on tumor cell type. Therefore, pharmacological treatments based on proapoptotic chemotherapy in association with autophagy regulators would benefit from a predictive in vitro screening of the target cell type.

Age-dependent modifications of Type 1 and Type 2 cytokines within virgin and memory CD4+ T cells in humans.

Several alterations in immune function and a concomitant progressive increase in pro-inflammatory status are the major characteristics of ageing process. Cytokines play a key role during ageing acting both in regulatory communication among cells and in effector activity during an immune response. The impact of age on intracellular Type 1 (IFN-gamma and TNF-alpha) and Type 2 (IL-4) cytokines, after stimulation with PMA/ionomycin, was determined in three CD4+ T subsets, i.e. CD95- CD28+ (virgin), CD95+ CD28+ (activated/memory), and CD95+ CD28- (effector/memory) from 47 subjects aged between 21 and 99 years. The percentage of IFN-gamma positive cells significantly decreased in virgin CD4+ subset both in old and nonagenarian subjects, as well as in activated/memory T cells from old in comparison with young subjects. The percentage of TNF-alpha positive cells significantly decreased in activated/memory CD4+ subset from old people. Regarding Type 2 cytokines, IL-4 positive cells significantly increased in activated/memory CD4+ subset from nonagenarians. On the whole our data indicate that: (1) different Type 1 and Type 2 cytokine-positive CD4+ T subsets are differently affected by ageing process; (2) activated/memory T cells appear to be the most affected subset; (3) a shift towards an increased role of Type 2 cytokines and a diminished role of Type 1 cytokines emerges with ageing.

Immunosenescence and infectious diseases.

Infectious diseases are major causes, with malignancies, of morbidity and mortality in the elderly. Increased susceptibility to infections may result from underlying dysfunction of an aged immune system; moreover, inappropriate immunologic functions associated with aging can determine an insufficient response to vaccines. Impairments of cellular, humoral and innate immunity in the elderly, contributing to increased incidence of infectious diseases, are discussed in this review.

The immune system in the elderly: III. Innate immunity.

The capability to cope with infectious agents and cancer cells resides not only in adaptive immune responses against specific antigens, mediated by T and B lymphocytes clonally distributed, but also in natural immune reactions. These innate defence mechanisms include chemotaxis, phagocytosis, natural cytotoxicity, cell interactions, and soluble mediators or cytokines. However, specific and natural immune mechanisms are always closely linked and interconnected, providing the primary defense against pathogens. The Authors discuss the main changes observed with advancing age in granulocytes and natural killer (NK) cell activity, in the expression and function of adhesion molecules, and in the pattern of cytokine production. Since phagocytic function is the primary mechanism through which the immune system eliminates most extracellular pathogenic microorganisms, analysis of this function is of clinical importance. Neutrophils from aged subjects often exhibit a diminished phagocytic capacity, as well as a depressed respiratory burst, notwithstanding an activated state. The activity of NK cells during aging has been studied extensively and different results have been reported. The most consistent data indicate an increase in cells with high NK activity with advancing age. Cells from healthy centenarians can efficiently kill target cells. This finding seems to suggest that innate immunity and in particular NK cell activity, is not heavily deteriorated with age. Conversely, a low NK activity is a predictor of impending morbidity. Immunosenescence is associated with increased expression of several cell adhesion molecules (CAM) resulting in an augmented capacity to adhere. Finally, also the cytokine network, responsible for differentiation, proliferation, and survival of lymphoid cells, undergoes complex changes with age. The main findings are a Th1 to Th2 cytokine production shift and an increased production of proinflammatory cytokines, which could explain many aspects of age-associated pathological events, such as atherosclerosis and osteoporosis.

Cell proliferation and apoptosis in the immune system in the elderly.

Loss of the cell proliferative capability and involution of tissues and organs are among the most important phenomena that characterize the aging process. Some of the aged-linked immune dysfunctions could be partly due to a dysregulation of apoptotic processes and to a lower responsiveness of aged lymphoid cells to activation and proliferation signals. The main changes in proliferative activity and cell death during aging and their impact on the process of immunosenescence are discussed. In fact, a very important function that has been suggested to deteriorate with age and to play a major role in the aging process is the capability of cells from aged subjects to respond to mitogenic stimuli and, consequently, to undergo cell proliferation. However, the cellular activation processes are very complex and the proliferative responses can follow different interconnected signal transduction pathways, and only some of them appear to be modified during age. Moreover, cell growth, immunosenescence, and longevity are strictly interconnected and deeply related to programmed cell death or apoptosis. The cellular equilibrium between cell survival and proliferation, on the one hand, and programmed cell death, on the other hand, seems to be unbalanced with advancing age, although in each type of immune cell it could be differentially modulated, resulting in a variety of clinicopathological consequences. Thus, cell proliferation and cell death are two physiologically active phenomena closely linked and regulated and a failure of these mechanisms determines profound dysregulations of cell homeostasis with major consequences in immune functioning and the onset of autoimmune diseases and cancer, whose incidence appears to be increased in the elderly.

The immune system in the elderly: I. Specific humoral immunity.

Profound and complex changes in the immune response occur during the aging process. Immunosenescence is reflected by a sum of disregulations of the immune system and its interaction with other systems. Many of the changes would appear to implicate age-related deficiencies of the immune responses. The term immunosenescence designates therefore a sort of deterioration of the immune function which is believed to manifest itself in the increased susceptibility to cancer, autoimmune disease, and infectious disease. Evidence has been accumulating from several studies which suggest an association between immune function and individual longevity. However, there are observations, especially in very old healthy people, that several immune functions are unexpectedly well preserved and substantially comparable to those observed in young subjects. These findings raise the question of whether the alterations that can be observed in the immune parameters of the elderly are a cause or a result of underlying disease processes. Moreover, studies on centenarians revealed a remodeling of the immune system rather than a deterioration, suggesting that the changes observed during immunosenescence do not correspond to immunodeficiency. The underlying mechanisms of these events are however still unclear. The purpose of the present review is to assess the status of research on the immunobiology of aging. In this first section, we focus attention on the B cell biology of aging. In clinical practice, the changes in humoral immune responsiveness and antibody-mediated defense mechanisms could greatly influence the incidence and outcome of bacterial infections and autoimmune diseases as well as the response to vaccines.

The immune system in the elderly: II. Specific cellular immunity.

Numerous changes occur in the immune system with advancing age, probably contributing to the decreased immunoresponsiveness in the elderly. These changes are often associated with important clinical manifestations such as increased susceptibility to infection and cancer frequently observed in the elderly population. Although both cellular and humoral immune responses are modified with advancing age, much of the decrease in immunoresponsiveness seen in elderly populations is associated with changes in T cell responses. The loss of effective immune activity is largely due to alterations within the T cell compartment which occur, in part, as a result of thymic involution. Substantial changes in both the functional and phenotypic profiles of T cells have been reported with advancing age. In fact, two prominent features of immunosenescence are altered T cell phenotype and reduced T cell response. One of the most consistent changes noted in T cells with advancing age is the decrease in the proportion of naive T cells with a concomitant increase in T cells with an activated/memory phenotype. In addition, there is evidence that the T cell population from aged individuals is hyporesponsive. The observed functional changes include decreased responsiveness to T cell receptor stimulation, impaired T cell proliferative capacity, a decline in the frequency of CD4+ T cells producing IL-2 and a decreased expression in IL-2 receptors. These latter findings probably explain the loss of proliferative capability of T cells from aged individuals. There is also evidence of a decrease in the early events of signal transduction, decreased activation-induced intracellular phosphorylation, and decreased cellular proliferative response to T cell receptor stimulation. The present review analyzes the main changes of the T cell compartment characterizing immunosenescence and discusses the possible mechanisms underlying these disregulations and their clinical implications.

Proliferating cell nuclear antigen expression is increased in small bowel epithelium in the elderly.

Although previous studies suggest that in aging animals the small intestine is in a hyperproliferative state, no information is currently available on the influence of age on the proliferation pattern of human small bowel enterocytes. The immunohistochemical expression of the proliferating cell nuclear antigen (PCNA), the villous height to total mucosal thickness ratio and the enterocyte height were evaluated in a panel of duodenal biopsy specimens obtained from 18 subjects aged less and 14 subjects aged more than 65 years. There was a significant positive correlation (P < 0.001) between age in years and percent of positive PCNA enterocytes both at the level of crypts (rs = 0.50) and villi (rs = 0.77). Moreover, the percentage of PCNA+ enterocytes was significantly higher in elderly versus adult subjects, both at the level of villi (6.5 vs 0%; P < 0.001) and of crypts (40.0 vs 23.7%; P < 0.01). No correlation was found between the percentage of PCNA + enterocytes and enterocyte height or villous height to total mucosal thickness ratio. Our results show that PCNA reactivity increases with advancing age both in crypts and villi. This abnormality of the proliferation pattern may explain the coexistence of normal morphology and impaired absorptive function in the elderly.

Levels of expression of CD52 in normal and leukemic B and T cells: correlation with in vivo therapeutic responses to Campath-1H.

The CD52 antigen is expressed on most normal and neoplastic lymphoid cells. The reshaped humanized IgG1 anti-CD52 monoclonal antibody (Campath-1H) has been used in the treatment of hemopoietic and non-hemopoietic diseases for its ability to induce lymphocyte depletion both in vitro and in vivo. Good activity has been shown in patients with chronic T and B cell leukemias, in particular T-prolymphocytic leukemia (T-PLL). However, the response to treatment is not uniform and this variability may depend on differences in the level of antigen expression on the leukemic cells. To test this hypothesis, we used quantitative flow cytometry to investigate the intensity of the expression of CD52 in 45 cases of lymphoid leukemia, 24 with B-cell chronic lymphocytic leukemia (CLL), 21 with T-PLL and 12 normal controls. Normal T lymphocytes expressed higher CD52 antigen than B lymphocytes (p < 0.005) and the antigen was also significantly higher in T-PLL compared to CLL (p < 0.001). Moreover, the differences in CD52 expression were somewhat higher in Campath-1H treated patients who responded than in non responders. Although other factors may play a role in the response to Campath-1H in vivo, the quantitative estimation of CD52 expression may provide a rationale for the greater response in T-PLL and help select those patients with a higher probability of responding to this therapy.

Altered lymphocyte antigen expressions in HIV infection: a study by quantitative flow cytometry.

To identify surface antigen changes that may contribute to the immune deficiency in infection with the human immunodeficiency virus (HIV), we quantified, by double-staining flow cytometry, the number of antigens of the main peripheral blood lymphocyte subsets from 30 HIV-positive persons and compared them with those of 19 HIV-negative healthy donors. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity values into numbers of antigen molecules per cell, measured as antibody binding capacity. The level of expression of different lymphocyte antigens in HIV-infected patients differs from that seen in normal blood lymphocytes. Some of these surface markers are decreased, whereas others are increased, and their expression is modulated depending on the specific cell subset considered. The expression of CD3, CD4, and CD8 on T lymphocytes is significantly decreased; moreover, CD3 is down-regulated on activated and nonactivated T lymphocytes and on CD4 and CD8 cells. In contrast, the expression of CD2 on T cells is significantly increased. Natural killer cells exhibit down-regulation of CD7, normal levels of CD8 and CD56, and overexpression of CD2. Our results also identified, for most of these antigens, quantitative differences in membrane expression according to different disease stages, as assessed by the CD4 T-cell count. Quantitative flow cytometry therefore may provide useful insights into the lymphocyte functional defects characterizing HIV infection.

Diamine oxidase plasma activities after treatment with heparin and jejunal morphometry in untreated coeliac disease.

Diamine oxidase plasma concentrations after treatment with heparin were measured and compared with the surface to volume ratio of jejunal biopsy samples assessed by a morphometric technique in patients with untreated and treated coeliac disease and in biopsied controls. As expected, enzyme activity was significantly lower in patients with untreated coeliac disease than in patients on a gluten-free diet and in biopsied controls. No difference was found between treated patients and biopsied controls. There was a significant overall correlation between plasma enzyme activity and surface to volume ratio of jejunal mucosa, although two untreated patients without an overt malabsorption syndrome but with a very low surface to volume ratio had normal enzyme activity. This study shows that in coeliac disease plasma diamine oxidase activity after treatment with heparin does not always mirror the extent of the jejunal lesions, particularly in those patients with minimal or unrelated symptoms who would benefit most from a valid screening test to identify their condition.

Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

AIMS: To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. METHODS: Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). RESULTS: CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. CONCLUSIONS: Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts.

Levels of expression of CD19 and CD20 in chronic B cell leukaemias.

AIMS: To investigate whether the antigen levels of the B cell lineage markers CD19 and CD20 can distinguish between normal and neoplastic B cells or characterise distinct expression patterns among the chronic B cell leukaemias. METHODS: Peripheral blood cells from 70 patients with B cell disorders and 17 healthy donors were analysed by quantitative flow cytometry. Direct immunofluorescence staining was performed with phycoerythrin conjugated CD19 and CD20 monoclonal antibodies. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values into number of antigen molecules/cell, expressed as antibody binding capacity (ABC). RESULTS: CD19 and CD20 ABC values in leukaemic B cells differed from those of normal blood B lymphocytes. The results identified distinct profiles of CD19 and CD20 expression in the various types of B cell leukaemias. In all leukaemias studied except hairy cell leukaemia (HCL), CD19 expression was significantly lower than the mean (SD) value in normal B cells (22 (7) x 10(3) molecules/cell), as follows: chronic lymphocytic leukaemia (CLL), 13 (7) x 10(3); B prolymphocytic leukaemia (B-PLL), 16 (9) x 10(3); splenic lymphoma with villous lymphocytes (SLVL), 15 (11) x 10(3); mantle cell lymphoma (MCL), 10 (7) x 10(3). In HCL there was strong CD19 expression (38 (16) x 10(3)). In contrast, the level of expression of membrane CD20 was higher than the mean (SD) value in normal B cells (94 (16) x 10(3) molecules/cell) in MCL (123 (51) x 10(3)); B-PLL (129 (47) x 10(3)); SLVL (167 (72) x 10(3)); and HCL (312 (110) x 10(3)); while it was significantly lower (65 (11) x 10(3)) in CLL compared with normal B cells and the other B cell leukaemias. CONCLUSIONS: Quantitative determination of CD19 and CD20 may provide useful diagnostic information for the study of B lymphoproliferative disorders.

Adhesion molecules on peripheral blood lymphocyte subpopulations in the elderly.

Adhesion molecules, such as CD49d, CD50 and CD62L, have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations are present in aged subjects, we studied, by means of triple colour whole blood immunostaining and multiparametric flow cytometry, the expression and intensity level (MFI) of these molecules on peripheral blood lymphocyte subpopulations from 23 healthy elderly subjects and 13 young controls. In the elderly a decrease in total peripheral blood lymphocytes bearing CD62L antigen was observed (39 +/- 13% vs 63 +/- 6% and 745 +/- 312/mm3 vs 1,393 +/- 407/mm3; p<0.001), whereas the numbers of lymphocytes expressing CD49d and CD50 antigens were comparable in aged and young subjects. In addition, CD50 and CD62L MFI values on total peripheral blood lymphocytes were higher in elderly than in young subjects (5.23 +/- 1.03 vs 4.18 +/- 0.44, p = 0.001 and 2.60 +/- 0.35 vs 2.21 +/- 0.40, p = 0.005 respectively) while the intensity expression of CD49d was unchanged. The percentages and absolute numbers of T and B lymphocytes expressing CD62L were decreased in elderly compared to young subjects (CD62L+CD3+: 43 +/- 15% vs 66 +/- 9% and 581 +/- 257/mm3 vs 1,028 +/- 418/mm3, p<0.001; CD62L+CD19+: 78 +/- 12% vs 90 +/- 4%, p < 0.005 and 103 +/- 64/mm3 vs 207 +/- 98, p < 0.001). A decrease in the proportion of CD62L bearing NK cells was also observed in the elderly (25 +/- 14% vs 46 +/- 24%, p<0.005), although their absolute number was unchanged. No significant differences were detected in the proportion of T, B and NK lymphocytes expressing CD49d and CD50 antigens and only the absolute numbers of B cells expressing these adhesion molecules were lower in elderly (CD49d+CD19+: 121 +/- 71/mm3 and CD50+CD19+: 107 +/- 73/mm3) compared to young donors (CD49d+CD19+: 248 +/- 112/mm3 and CD50+CD19+: 235 +/- 120/mm3, p < 0.001). Moreover, the intensity of adhesion molecule expression was differentially modulated in the elderly depending on the specific lymphocyte cell population considered. The densities of CD49d, CD50 and CD62L antigens on B and NK lymphocytes from the two age groups were not different; on the contrary, T lymphocytes from elderly donors exhibited increased CD49d (1.69 +/- 0.09 vs 1.62 +/- 0.07, p < 0.05), CD50 (4.98 +/- 1.16 vs 3.77 +/- 0.46, p < 0.001) and CD62L (2.26 +/- 0.38 vs 1.99 +/- 0.37, p < 0.05) MFI values compared to young donors.

The impact of HIV infection on lactose absorptive capacity.

Forty HIV-infected adult patients at different disease stages and 44 healthy volunteers were evaluated for lactose malabsorption using the hydrogen breath test after 20 g lactose ingestion. All subjects were previously tested for breath hydrogen (H2) excretion after 12 g lactulose ingestion. The presence of intestinal superinfections, gastrointestinal symptoms and the intensity of clinical intolerance after lactose load were accurately searched in each patient. The cumulative H2 excretion after lactulose did not significantly differ between the different groups studied. The prevalence of lactose malabsorption turned out to be significantly higher (P < 0.001) in HIV-infected patients (70%) than in controls (34%). Moreover, in patients in more advanced disease stages the degree of lactose malabsorption was significantly greater than in patients at earlier disease stages, who did not differ from healthy volunteers. Furthermore the degree of lactose intolerance was significantly greater (P < 0.001) in symptomatic patients than in those without intestinal symptoms and in healthy volunteers, while no significant difference was observed between these latter groups. The results here demonstrate the negative impact of HIV infection on lactose absorptive capacity in adult patients, particularly marked in more advanced stages of the disease, suggesting that, in addition to the presence of the virus alone, other factors may contribute to determine the enterokinetic alterations responsible for lactase deficiency.

Recurrence of Hodgkin's disease after a 28-year disease-free interval. A case report.

The authors describe a case of nodular-sclerosing Hodgkin's disease, originally observed in clinical stage III B and treated by systemic chemotherapy, with relapse after a 28-year disease-free interval. As far as we know, the length of remission in this case is second only to that of a case recently described by Hung and co-workers. Therefore, although according to data in the literature relapse after a very long disease-free interval is more frequently observed in patients with stage I A and II A disease, it may occur also in cases with symptomatic, widespread disease. This phenomenon, while reflecting the complexity of relations between the different factors (histologic subtype, clinical stage, host vs tumor defenses, therapy, etc.) that variously affect the clinical course and outcome, underlines the need for cautiousness when assessing the ultimate prognosis of individual cases of Hodgkin's disease.

Epididymal lymphoma. A case report.

Primary epididymal lymphoma is an unusual observation. Only 2 cases of non-Hodgkin's lymphoma of the epididymis have been previously reported. We describe the clinical and pathologic features and management of a primary high-grade malignant lymphoma of the epididymis in which a tentative diagnosis of lymphoma was made on the basis of cytologic examination and immunochemical staining of the material obtained from an aspiration needle biopsy.