Outcomes after laparoscopic Nissen fundoplication are not influenced by the pattern of reflux.

The purpose of this study was to compare the outcomes of patients with different types of gastroesophageal reflux disease (upright, supine, or bipositional) after laparoscopic Nissen fundoplication and determine if patients with upright reflux have worse outcomes. Two hundred and twenty-five patients with reflux confirmed by 24-h pH monitoring were divided into three groups based on the type of reflux present. Patients were questioned pre- and post-fundoplication regarding the presence and duration of symptoms (heartburn, regurgitation, dysphagia, cough and chest pain). Symptoms were scored using a 5-point scale, ranging from 0 (no symptom) to 4 (disabling symptom). Esophageal manometry and pH results were also compared. There was no statistically significant difference in lower esophageal sphincter length, pressure or function between the three groups. There was no significant difference in any of the postoperative symptom categories between the three groups. The type of reflux identified preoperatively does not have an adverse effect on postoperative outcomes after Nissen fundoplication and should not discourage physicians from offering antireflux surgery to patients with upright reflux.

Calcein-acetyoxymethyl cytotoxicity assay: standardization of a method allowing additional analyses on recovered effector cells and supernatants.

Cytotoxicity assays provide an in vitro evaluation of the lytic activity of NK and T cells against tumors or transformed cells. However, none of these methods allow the recovery of cells or supernatants after the assay. We standardized a microcytotoxicity test using calcein-acetoxymethyl (calcein-AM) dye that requires very small quantities of cells while maintaining the same sensitivity as the traditional (51)Cr assay. The assay is applicable to resting as well as activated human effector cells and uses different targets such as human cell lines that are adherent or growing in suspension and resistant or sensitive. The most important feature of the method is the possibility of recovering cells and supernatants for additional analyses such as phenotyping and evaluation of soluble factors.

Limitations of randomized clinical trials for evaluating emerging operations: the case of lung volume reduction surgery.

Although unanswered questions remain, scores of observational studies and several small randomized clinical trials (RCTs) indicate that lung volume reduction surgery (LVRS) offers safe and effective palliation for a relatively well defined subset of patients with advanced emphysema. Nonetheless, Medicare and other insurers stopped reimbursement for the procedure. Subsequently, two multicenter RCTs on LVRS, the National Emphysema Treatment Trial (NETT) and the Overholt-BlueCross Emphysema Surgery Trial (OBEST), were launched with the stipulation that the procedure would not be paid for outside these trials. Thus access to LVRS has been denied to patients who could benefit but do not wish to participate in an RCT. Emerging operations, unlike new drugs or devices, pass through evolutionary changes and frequently fail to produce data that meet the scientific rigor required by randomized studies. In such a setting, the observational approach is more appropriate. Indeed, almost all operations in the present surgical armamentarium have been evaluated and have evolved through observational studies without the use of RCTs. By the time new operations are standardized and qualify for RCTs, benefits for certain patients may be demonstrated and randomization could involve unacceptable health hazards. Patients from this population should be offered the choice between participating in RCTs and having the operation outside the study. Imposition of financial restrictions that bars access to a therapy with known benefit is a questionable practice.

Different IL-8 production by T and NK lymphocytes in elderly subjects.

A gradual decline in the functional activity of the immune system is described with advancing age. The adaptive immune system seems the most severely affected, but some age-associated modifications also occurs in NK cells. Several studies investigated the age related changes of cytokine production, while little is known about chemokines, whose importance in regulating immune-response becomes even more evident. In this study we investigated whether the ability of T lymphocytes and NK cells to produce IL-8, either spontaneously or after activation, respectively with anti-CD3 monoclonal antibody or interleukin 2 (IL-2) was affected by age. We demonstrated that: (a) T lymphocytes and NK cells spontaneously produced detectable amounts of IL-8; (b) anti-CD3 stimulation of T lymphocytes significantly increased IL-8 production and the increment was more evident in the nonagenarian subjects; (c) similarly, IL-2 stimulation of NK cells rose the production of IL-8 but the amount produced by the old was lower than the one produced by the young group. Because of the co-stimulatory role of chemokines on NK responses and given the demonstrated importance of NK cells in defence against viral infections, the decreased production of IL-8 can be involved in the defective functional activity of NK cells from old subjects.

Interleukin-12 induces efficient lysis of natural killer-sensitive and natural killer-resistant human osteosarcoma cells: the synergistic effect of interleukin-2.

Previously we demonstrated that some osteosarcoma cell lines varied greatly in their susceptibility to natural killer (NK) cell lysis in vitro. The expression of CD54 and CD58 adhesion molecules on their surface appeared to influence their vulnerability, and the tumour necrosis factor-alpha (TNF-alpha)-induced positive modulation of CD54 increased osteosarcoma susceptibility in vitro. This study investigated whether peripheral blood mononuclear cells from normal healthy donors could be activated by interleukin (IL)-12 and IL-2, separately or in combination, to lyse osteosarcoma cell lines in vitro, as evaluated by using a microcytotoxicity test. In addition, we analysed (by flow cytometry) whether this function correlated with modifications of the CD2, CD11a, CD11b and CD18 molecules, which are involved in the adhesion of effector cells to the counter-receptors (CD54 and CD58) on osteosarcomas. This study demonstrates that incubation with IL-12 and/or IL-2 triggered NK cell cytolytic activity against osteosarcoma targets and that cytolytic activity was enhanced to a greater extent when lymphocytes were incubated simultaneously with a combination of IL-12 and IL-2. The density of CD18 and CD2 molecules involved in NK adhesion was also up-modulated following cytokine incubation. These changes in the density of adhesion molecules can be involved in the increased lytic activity of effector lymphocytes and in the modification of their binding capacity to osteosarcoma target cells.

Chemokine production by peripheral blood mononuclear cells in elderly subjects.

The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections. In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Ialpha, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin. Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Ialpha and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lalpha. In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in 'specific' immune response during ageing. The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.

NK binding capacity and lytic activity depend on the expression of ICAM-1 on target bone tumours.

Osteosarcoma cell lines are differently lysed by natural killer (NK) lymphocytes. A critical step in the lytic process is the recognition and attachment of effector to target cells. To determine binding capacity and lytic activity of NK cells, we investigated the distribution and role of ICAM-1, 2 and 3 on two osteosarcoma cell lines (HOS and Saos-2) in basal conditions and after TNFalpha treatment. Modulation of ICAM-1 after TNFalpha treatment modified the binding capacity of NK cells to osteosarcoma target cells. This modulation process appears to play a critical role in determining the susceptibility of these cells to NK-mediated lysis.

Vitamin D, thyroid hormones and muscle mass influence natural killer (NK) innate immunity in healthy nonagenarians and centenarians.

Increasing evidence has demonstrated that the immune system closely interacts with other physiological systems, whose communications are mediated by circulating cytokines and hormones. The aim of our study was to test whether the number and cytolytic activity of NK cells in a group of relatively healthy Italian nonagenarians and centenarians were affected by the modifications of endocrine, metabolic and functional parameters that occur during ageing. Because of the extreme age of the study population, a cross-sectional analysis was performed. This study revealed that the group of oldest subjects with the highest number of NK cells and the best preserved cytolytic function also presented a preserved metabolism of thyroid hormones and vitamin D and integrity of muscle mass. In fact, the NK cell number and/or cytolytic activity of healthy subjects > 90 years old was positively associated with serum levels of vitamin D, while T3, FT4, i-PTH hormones and lean body mass were associated only with NK cell number. In conclusion, our results stress the paramount importance of nutritional evaluation in the clinical assessment of elderly people.

Age-dependent decreases of NK cell phosphoinositide turnover during spontaneous but not Fc-mediated cytolytic activity.

The progressive increase in the number of peripheral NK cells found in the elderly does not correlate with a corresponding increase in lytic activity. On the contrary, a decreased function of circulating NK cells purified from old subjects was observed on a per cell basis. Most of the studies on NK cells have focused on late events such as lytic activity. In view of this, little is currently known about the modification of the early signalling pathways of NK cells in elderly people. This study investigated whether the modification of NK lytic activity could be related to differences in the metabolic pattern of activation of these cells in the elderly. NK cells were negatively purified by immunomagnetic depletion from the peripheral blood of selected old and young healthy subjects. Hydrolysis of inositol phospholipids was measured following incubation with K562 target cells and/or CD16 mAb for different times. Our data show that there is a pronounced age-related decrease in the ability to generate total inositol monophosphates and, particularly, inositol trisphosphates by NK cells following K562 stimulation (spontaneous cytolytic activity) together with an attenuated and delayed hydrolysis of phosphatidylinositol bisphosphate, while phosphoinositide turnover is preserved following Fc triggering (antibody-dependent cell-mediated cytotoxicity). These results confirm that, also in old subjects, different biochemical pathways of activation are involved in NK cells when target or antibody-mediated triggering occurs and may aid the development of experimental and therapeutic strategies to counteract declines in cell mediated immune functions associated to advancing age.

Natural immunity and bone and muscle remodelling hormones in the elderly.

Increasing evidence has demonstrated that the immune system is closely integrated with two other physiological systems: endocrine and nervous. They communicate through circulating humoral factors such as cytokines, hormones and neurotransmitters. We undertook a cross-sectional analysis in a group of elderly subjects over 90 years to demonstrate that a functional relationship exists among the number and cytolytic activities of NK cells, bone and muscle remodelling hormones, anthropometric parameters and physical ability. Peripheral blood samples collected from 62, 90-106 years-old subjects underwent biochemical (bone and muscle remodelling hormone levels) and immunological determinations (Natural Killer cell distribution and activity), anthropometric and functional assessment. Significant associations were found among NK cell number and cytolytic activity and serum concentrations of vitamin D, anthropometric parameters, while functional independence in daily activity was only associated with NK cell number. In general a high level of physical ability was correlated with preserved body stores and vitamin D levels. In conclusion, our results stress the importance of nutritional evaluation in the clinical assessment of elderly people. The magnitude of the NK immune response, which constitutes the first line of defence against infected and neoplastic cells, is best preserved in oldest-old people with the best hormonal parameters and nutritional measures.

TNF-alpha but not IL-1 and IL-6 modifies the susceptibility of human osteosarcoma cells to NK lysis.

TNF-alpha-treated osteosarcoma cells have an enhanced susceptibility to NK lysis which mostly depends on the increased expression of CD54 molecules. Since IL-1 and IL-6 share overlapping biological properties with TNF-alpha, we investigated whether the treatment of osteosarcoma cells with these cytokines could modify their susceptibility to NK lysis and whether these modifications were related to a different distribution of CD54, CD56 and CD58 molecules. We demonstrated that the expression of CD54 and CD58 on osteosarcomas correlated positively with the susceptibility to NK lysis and that this susceptibility was enhanced by TNF-alpha treatment but not by IL-1 and IL-6 stimulation.

Expression of beta-galactoside alpha 2,6-sialyltransferase does not alter the susceptibility of human colon cancer cells to NK-mediated cell lysis.

The extent of processing of N-linked oligosaccharides and the sialylation of the target cell membranes has been positively correlated with resistance to lysis mediated by NK cells, but a conclusive evidence has never been reached. Colon cancer tissues express an increased activity of beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1, alpha 2,6ST), which catalyzed the addition of sialic acid in alpha 2,6-linkage to Gal beta 1,4GlcNAc (N-acetyllactosamine) sequences of glycoprotein N-linked chains. The resulting increased level of membrane alpha 2,6-sialylation appears to be related with a more invasive behavior of cancer cells. This phenomenon may depend on a decreased sensitivity of colon cancer cells to NK cells. To obtain conclusive evidence on the role played by sialylation of N-linked chains in determining the target cell susceptibility to NK-mediated lysis, human colon cancer cell lines not expressing sialyltransferases acting on N-linked chains were transfected with a rat alpha 2,6ST cDNA. Stable transfectants expressed different levels of alpha 2,6ST activity, were reactive with the Sambucus nigra lectin, specific for alpha 2,6-linked sialic acid, and compared with control transfectants, showed a remarkable decrease in the number of unsubstituted Gal beta 1,4GlcNAc terminal sequences. The NK susceptibility of these clones was found to be identical to that of control transfectants, either when unstimulated- or IL-2-stimulated lymphocytes were used as effectors. Neuraminidase treatment of target cells does not result in significant changes to NK susceptibility. Our data demonstrate that sialic acid alpha 2,6-linked to N-linked chains of target cell glycoproteins does not play a major role in recognition of the target by human NK cells.

Human osteosarcoma cell susceptibility to natural killer cell lysis depends on CD54 and increases after TNF alpha incubation.

Osteosarcoma cell lines vary widely in their susceptibility to natural killer (NK) cell lysis in vitro although it is still unclear why this occurs. In this study we investigated the expression of some cell adhesion molecules on osteosarcomas to determine which of these can modify the susceptibility to NK lysis and we also attempted to modulate the cytolytic susceptibility of these targets with TNF alpha. We found that osteosarcoma lysis induced by NK cells correlates with different expression of the CD54 adhesion molecule on osteosarcomas and the increased susceptibility after TNF alpha treatment mostly depends on the expression of CD54 molecules on target cells.

Perforins in human cytolytic cells: the effect of age.

The ageing process is associated with a progressive increase in the number of circulating NK cells, together with a decreased lytic activity per cell. A similar decrease in activity was also found for CD8 lymphocytes. Cytotoxic T- and NK cells express cytoplasm granules containing cytolytic effector molecules (as perforins, studied here) which can recognize and destroy damaged, infected and/or mutated target cells. To investigate whether an altered distribution of perforins in cytolytic cells or a reduced number of cytolytic cells producing perforins underlies decreased cytolytic activity with advancing age, perforin expression was assessed at the single cell level in T- (CD4 and CD8) and NK (CD16) peripheral blood lymphocytes from elderly subjects by flow cytometry. Perforin distribution at the cellular level in CD8+ and CD16+ cell cytoplasm suggested a similar distribution during ageing and a similar number of cells producing perforins. In addition, perforin utilization was maintained in the generation of cytolytic activity against K562 target cells and perforin synthesis in culture following activation was unabated. These data stress the importance of other factors, such as defective signal transduction for granule exocytosis, that may account for the different pattern of lytic activity found in aged people.

Epithelial marker genes are expressed in cultured embryonic rat lung and in vivo with similar spatial and temporal patterns.

Explants of embryonic lung are often used to characterize lung growth, bronchial tree pattern, and cell differentiation. Most investigators culture lungs for 3-7 days in defined media lacking, e.g., added growth factors or hormones. If growth and differentiation are comparable to that in vivo, these cultures show considerable promise for identifying developmental regulatory molecules and target genes, and for elucidating molecular responses. We used in situ hybridization and RT-PCR to compare times and sites of expression of mRNAs of six epithelial genes in cultured and uncultured fetal rat lungs. These genes, expressed in distal lung of adult rats, are surfactant proteins (SP) A, B, and C; LAR, a receptor-type tyrosine phosphatase; Clara cell secretory protein (CC10, CCSP); and T1alpha. SP-A, SF-B, LAR, and CC10 are expressed by both Clara and Type II cells in adult animals. SP-C and T1alpha are unique markers for Type II and Type I cells, respectively. SP-C, LAR, and T1alpha are expressed before the lung is explanted (Day 13.5); SP-A, -B, and CC10 mRNAs are first detected later. The onset of expression is similar in vivo and in vitro. Although the patterns of expression differ for each mRNA, their sites of expression in culture match those in vivo relative to the bronchial tree. The explanted embryonic lung appears to be an excellent experimental model.

Lytic activity of IL-2 and IL-12 stimulated NK cells against HOS osteosarcoma cell line.

NK cells are CD16, CD56 positive lymphocytes that spontaneously lyse tumor or virus infected cells. In this study we investigated whether IL-2 and/or IL-12 stimulated NK cells increased their lytic efficiency against HOS osteosarcoma cell line. Our results demonstrate that both 18 hour and 5 day incubation times enhanced the lytic activity of human PBL against HOS and K562 target cells and that IL-12 appears to be more efficient than IL-2 in augmenting NK cytotoxicity.