Non-Contact Universal Sample Presentation for Room Temperature Macromolecular Crystallography Using Acoustic Levitation.

Macromolecular Crystallography is a powerful and valuable technique to assess protein structures. Samples are commonly cryogenically cooled to minimise radiation damage effects from the X-ray beam, but low temperatures hinder normal protein functions and this procedure can introduce structural artefacts. Previous experiments utilising acoustic levitation for beamline science have focused on Langevin horns which deliver significant power to the confined droplet and are complex to set up accurately. In this work, the low power, portable TinyLev acoustic levitation system is used in combination with an approach to dispense and contain droplets, free of physical sample support to aid protein crystallography experiments. This method facilitates efficient X-ray data acquisition in ambient conditions compatible with dynamic studies. Levitated samples remain free of interference from fixed sample mounts, receive negligible heating, do not suffer significant evaporation and since the system occupies a small volume, can be readily installed at other light sources.

Use of translation cards to increase communication with non-English-speaking families in the NICU.

Parents of infants in the neonatal intensive care unit report that communication with staff helps to relieve the many stresses they face. Particularly challenging for NICU nurses, however, is communication with non-English-speaking families. To improve this communication at Vanderbilt Children's Hospital in Nashville, illustrated cards have been developed to translate phrases commonly used in the nursery into foreign languages. Three different sets of cards provide translations in Spanish, Kurdish, and Serbo-Croatian (the languages most commonly encountered at this hospital), and a fourth set, in English, is used for individuals with hearing impairments. These cards aid the bedside nurse in giving a basic report and in encouraging parental participation in the infant's care. Nurses who have used the cards report that parents respond to the questions and seem to appreciate the attempt to communicate.

Characterization of tumor vaccines during product development.

The term tumor vaccines encompasses a wide variety of diverse agents capable of interacting with the immune system to produce local inflammation, delayed type hypersensitivity reaction and/or tumor regression and, hopefully, a therapeutic effect. These vaccines may be grouped into the following general areas: (1) Cell-based vaccines such as manipulated tumor cells, activated peripheral blood or bone marrow-derived lymphocytes, dendritic cells or other antigen presenting cells (APC) and gene-modified tumor cells or other cells engineered to express cytokines, growth factors or tumor antigens. (2) Antigen preparations, such as synthetic peptides, purified antigens and tumor cell lysates. (3) Viral and plasmid vectors expressing therapeutic genes. (4) Liposome containing antigen, peptides, plasmids encoding tumor antigens. While no tumor vaccine has been licensed by the FDA, numerous clinical trials are ongoing and some products have advanced to Phase III pivotal stages of development. However, as with many novel products, major regulatory and scientific issues associated with clinical use of tumor vaccines remain to be addressed. In this paper, we address issues associated with different types of tumor vaccines and provide recommendations for the characterization of these vaccines at various stages of development.

Presence of CD4+ T suppressor cells in mice rendered unresponsive to tumor antigens by intravenous injection of irradiated tumor cells.

We used in vivo and in vitro assays to determine whether suppressor cells are generated in mice rendered unresponsive to tumor-specific antigens by intravenous (i.v.) injection of non-replicating tumor cells. The results show that a single i.v. injection of 2 x 10(7) irradiated P815 tumor cells resulted in the induction of a state of specific unresponsiveness to tumor-associated antigens, as revealed by the inability of the treated mice to achieve immunologically-mediated regression of an intradermal P815 tumor containing C. parvum, or to generate effector T cells capable of causing rejection of a P815 tumor in T-cell-deficient (T x B) test recipients. Failure to respond to tumor antigens was associated with the presence in spleen of CD4+ T cells capable, on passive transfer, of suppressing adoptive T-cell-mediated tumor regression in T x B recipients. However, the same CD4+ suppressor cells failed to inhibit the generation of tumor-specific cytotoxic T lymphocytes (CTL) in vitro. On the contrary, spleen cells from mice made unresponsive by i.v. injection of tumor cells were primed to generate CTL in response to tumor antigens in vitro. Taken together, our results suggest that unresponsiveness induced by i.v. injection of tumor antigens is an active process mediated, at least in part, by CD4+ T suppressor cells, and that these cells coexist in the spleen with antigen-primed effector T cells with a capacity to generate tumor-specific CTL when released from suppression in vitro.

Evidence inconsistent with a role for tumor necrosis factor in tumor allograft rejection.

Since it has been suggested that tumor necrosis factor is involved in organ allograft rejection, experiments were undertaken to determine whether it is involved in the rejection of allogeneic P815 tumor cells growing as ascites in the peritoneal cavities of mice. The results show that biologically active TNF was not detectable in serum or ascites fluid of mice during either growth or vigorous rejection of the tumor. On the other hand, mice in the process of rejecting their ascites tumor displayed a greatly enhanced capacity to produce TNF in the peritoneal cavities and systemically in response to an injection of endotoxin i.p. These results show that the immune response to the tumor was associated with the priming of host cells for increased TNF production both locally and systemically in response to an appropriate stimulus that was not supplied during the generation and expression of immunity. Additional evidence against a role for TNF in ascites tumor allograft rejection is seen in the finding that i.p. administration of anti-TNF antibodies failed to interfere with elimination of allogeneic tumor cells, even though the antibodies were given repeatedly before and during the rejection process. Taken together, these results are inconsistent with the view that TNF is involved in the effector stage of the anti-allograft response, even though cells at the site of rejection are primed to produce large quantities of TNF in response to endotoxin.

The accumulation of bactericidal lipids in staphylococcal abscesses.

Abscesses were generated in the peritoneal cavity of mice by the inoculation of 10(9) staphylococci. Abscess weight increased rapidly, reaching about 200 mg by the fourth day; for the next 60 days, abscess weight increased only slightly. The amount of total lipid increased during abscess development, attaining a peak level of about 19 mg per abscess at 7 days before decreasing. Almost all of this lipid resulted from the accumulation of neutral lipids. The small increases seen in the phospholipid and glycolipid fractions could be accounted for through the accumulation of host cellular elements in the abscess. Leucocytes containing cytoplasmic lipid droplets were first seen 4-12 h after infection and these cells were widely scattered around the periphery. During the next 2 days, the number of cells with lipid droplets increased markedly and lipid droplets were also found in the deeper portions of the abscesses. Although lipid droplets were found subsequently throughout the abscess, the greatest amounts always occurred in the leucocyte zone immediately proximal to the connective tissue capsule. During abscess development, the bactericidal activity also increased rapidly, reaching a maximum by the seventh day and declining thereafter.

Identification of borderline personality disorder with the NIMH Diagnostic Interview Schedule.

No lay-administered interviews are currently available to identify persons with borderline personality disorder. The authors studied 79 subjects with the NIMH Diagnostic Interview Schedule (DIS), a lay-administered interview, and the Diagnostic Interview for Borderline Patients (DIB) and used the results to construct a DIS-based diagnostic index to identify borderline personality disorder. Using the clinician-administered DIB as the diagnostic standard, the authors found that the DIS borderline index had a sensitivity of 85.7%, a specificity of 86.2%, and a kappa of 0.67. The DIS borderline index is a promising extension of the DIS that will facilitate studies of borderline personality disorder in clinical and community settings.

The antimetastatic function of concomitant antitumor immunity. II. Evidence that the generation of Ly-1+2+ effector T cells temporarily causes the destruction of already disseminated tumor cells.

Progressive growth of the P815 mastocytoma in an immunocompetent host evokes the generation of an antitumor immune response that can be measured in terms of the production of cytolytic Ly-1+2+ T cells in the draining lymph node and spleen. This immunity, designated concomitant immunity, is present on day 6 of tumor growth, peaks on day 9, and decays progressively thereafter. It fails to develop in mice made T cell deficient by thymectomy and lethal whole-body gamma-radiation, and reconstituted with syngeneic bone marrow cells (TXB mice). Employment of a mouse survival assay, capable of enumerating metastatic P815 cells in cell suspensions, showed that the P815 tumor metastasizes to the draining lymph node and spleen at the same rate in normal and TXB mice for the first 6 days of growth of an intradermal P815 tumor. By day 6 of tumor growth there were approximately 10(3) P815 cells in the draining lymph node in both types of mice. However, during the generation of concomitant immunity between days 6 and 9, the number of metastatic P815 cells in the draining lymph nodes and spleens of normal tumor-bearing mice declined by nearly 90%. After day 12, however, the number of tumor cells in the nodes and spleens increased concordantly with the decay of concomitant immunity. These findings, together with the demonstration that T cell-deficient mice failed to restrain the number of metastatic P815 cells in the draining lymph node and spleen, suggest that concomitant immunity is an important defense mechanism against the development of systemic disease. Additional evidence consistent with this interpretation was provided by studies which showed that adoptive immunization with spleen cells from concomitant immune donors significantly prolonged the median survival time of TXB tumor-bearing mice by destroying a substantial proportion of P815 tumor cells already seeded in the draining lymph node. Adoptive immunization also delayed the appearance of metastatic tumor cells in the spleen.

The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells.

Controversy exists over a direct effect of 1,25(OH)2D3 on PTH secretion. To investigate the possibility that the suppressive effect of 1,25(OH)2D3 on PTH secretion may be demonstrable in 1,25(OH)2D3-depleted tissue and/or after prolonged periods of exposure to 1,25(OH)2D3, primary monolayer cultures of bovine parathyroid cells were established in 1:1 DMEM/Ham's F-12 media supplemented with 2% calf serum but not 1,25(OH)2D3. Ionized calcium was maintained at 1.0 mM. Experiments were performed on 4-day-old culture cells. PTH concentration was measured using both a mid-region/carboxyl and an amino-terminal PTH antisera. 1,25(OH)2D3 at a concentration of 0.1 ng/ml suppressed PTH secretion by 32 +/- 7% after 48 hours. High calcium concentration (2.0 mM) suppressed PTH secretion by 37 +/- 10% and this effect was not additive over that of 1,25(OH)2D3. PTH secretion rate recovered fully 48 hours after normalization of the external calcium concentration but not after the removal of 1,25(OH)2D3. It is concluded that 1,25(OH)2D3 directly suppresses PTH secretion by monolayer culture of bovine parathyroid cells.

Pancreatic polypeptide in Pima Indians: the influence of obesity and diabetes.

The influence of obesity and diabetes on circulating pancreatic polypeptide (PP) levels was studied in 62 Pima Indians and 22 caucasians. Plasma PP was determined in the fasting state and after a standardized test meal. Fasting and the postprandial PP responses were not significantly different among the Pima Indians whether nonobese, obese, or diabetic. However, their concentrations were significantly higher both fasting and postprandially compared to those of caucasians. In both groups the postprandial PP response was positively correlated with the fasting level. Fasting and postprandial PP levels positively correlated with age in Pimas.

Ly 1+2- suppressor T cells down-regulate the generation of Ly 1-2+ effector T cells during progressive growth of the P815 mastocytoma.

It has been shown that progressive growth of the P815 mastocytoma in its semisyngeneic B6D2 host results, between days 6 and 9 of tumour growth, in the generation of Ly 1-2+ T cells which are capable, on passive transfer, of causing the regression of an established tumour in gamma-irradiated recipients, and of T cells which are capable of lysing P815 tumour cells in vitro. After 9 days of tumour growth, these effector T cells are progressively lost; this is associated with the progressive acquisition of Ly 1+2- suppressor T cells which are capable of inhibiting the expression of passively transferred immunity against an established tumour in T-cell deficient recipients. The results are consistent with the hypothesis that this P815 mastocytoma grows progressively, in spite of its immunogenicity, because it evokes the generation of suppressor T cells before enough effector T cells are generated to reject it.

Specificity of the T cells that mediate and suppress adoptive immunotherapy of established tumors.

This study was designed to investigate the specificity of the T cells that express and suppress antitumor immunity in a model of adoptive immunization against established tumors. Results obtained with the P815 mastocytoma, L5178Y lymphoma, and P388 lymphoma showed, in agreement with previous findings from this laboratory, that an intravenous infusion of splenic T cells from immunized mice can cause the regression of a tumor growing in T-cell-deficient mice. So far as specificity of adoptive immunity is concerned, reciprocal passive transfer experiments with these three tumors revealed that T cells from donor mice immunized against the P815 tumor are not capable of causing regression of the P388 tumor or L5178Y tumor, even if both the P815 and L5178Y tumors are growing in the same host. Similarly splenic T cells from mice immunized against the P388 tumor or L5178Y tumor had no effect on growth of the P815 tumor. Suppression of adoptive immunity was also specific, in that passively transferred suppressor T cells from mice bearing a progressive P815 tumor were capable of suppressing adoptive T-cell-mediated regression of the P815 tumor, but not the P388 tumor growing in T-cell-deficient recipients. Reciprocally, P388 suppressor spleen cells from mice bearing a progressive P388 tumor prevented adoptive T-cell-mediated regression of the P388 tumor, but not the P815 tumor. The results indicate, therefore, that the T cells from immunized mice that mediate adoptive antitumor immunity and the T cells from tumor-bearing mice that suppress the expression of this immunity are specific for the tumor that evokes their generation.

Adoptive immunization against an established tumor with cytolytic versus memory T cells. Immediate versus delayed onset of regression.

Intradermal injection of an admixture of P815 tumor cells and Corynebacterium parvum results in the emergence of a tumor that grows progressively for 9-10 days and then undergoes complete regression. Tumor regression is preceded by a cytolytic T cell response in the spleen that peaks on day 10 and then undergoes progressive decay until days 15-16 when cytolytic T cells can no longer be detected. Passive transfer of 10-day or 30-day spleen cells to T-cell-deficient recipients bearing a 4-day tumor resulted in complete tumor regression. However, whereas passively transferred 10 day spleen cells caused the onset of tumor regression within 2 days, passively transferred 30-day spleen cells did not cause tumor regression until after a 6-8-day delay. Again, the antitumor function of 10-day spleen cells could be eliminated by treatment with cyclophosphamide and vinblastine sulfate, whereas 30 day spleen cells were resistant to both agents. These results indicate that 10-day spleen cells are physiologically different from 30-day spleen cells. The results are consistent with the interpretation that passively transferred 10-day spleen cells cause rapid onset of tumor regression because they are cytolytic T cells and have an immediate capacity to destroy the tumor. In contrast, 30-day spleen cells are helper or memory T cells with no capacity at the time of transfer to destroy the tumor.

A new in vitro model for studies of pancreatic polypeptide secretion and biochemistry.

A new model tissue (pseudoislet) is described for studies of pancreatic polypeptide (PP) secretion and biochemistry. It consists of islet-like aggregates of canine pancreatic endocrine cells which are formed and maintained on tissue culture. Immunocytochemical staining revealed that pseudoislets prepared from the duodenal end of the pancreas contained a predominance (40-60%) of F cells (the PP secreting cell). Also present were 10-25% exocrine cells and an equal proportion of A, B and D cells. Several studies were conducted to characterize the pseudoislets' capacity to secrete PP. Basal rates of PP release and the concentration of PP per pseudoislet remained constant during four weeks of culture. Stimulation at weekly intervals by carbachol (0.1 mM) resulted in a stable secretory rate for 2 weeks, that declined progressively at weeks 3 and 4. When studied in a perfusion system, carbachol-stimulated PP release occurred in a biphasic pattern, similar to the well-recognized biphasic release of insulin from perifused rat islets. Dose-response curves of four cholinergic agonists revealed clear differences in secretagogue activity. Acetylcholine and methacholine were found to be equipotent, followed in order of potency by carbachol and bethanechol. These histologic and secretory data show that canine pseudoislets are healthy tissues composed of a high proportion of F cells which secrete PP in response to cholinergic stimulation. The data suggest that the cultured canine pseudoislet model provides an excellent system useful in studies of PP secretion and biosynthesis.

T cell-mediated immunosuppression as an obstacle to adoptive immunotherapy of the P815 mastocytoma and its metastases.

Progressive growth of the P815 mastocytoma in semisyngeneic mice evokes the generation of a T cell-mediated mechanism of immunosuppression that inhibits the capacity of passively transferred, tumor-sensitized T cells from regressing this tumor in recipient mice. This conclusion is based on two findings: (a) that it is possible to demonstrate adoptive T cell-mediated regression of established tumors, but only if the tumors are growing in T cell-deficient recipients, and (b) that adoptive T cell-mediated regression of tumors in these recipients can be inhibited by the infusion of splenic T cells from T cell-intact, tumor-bearing donors. The results of additional experiments designed to measure the effect of decreasing the number of suppressor cells and the time that they are infused, relative immune cells, indicate that the function of suppressor cells in this model is to inhibit the replication of passively transferred immune T cells. The results obtained with the P815 mastocytoma are similar to those obtained previously with a chemically induced fibrosarcoma. They show, in addition, that passively transferred immune cells are capable of destroying already seeded metastases in T cell-deficient recipients.

Mechanisms of anti-tumor action of Corynebacterium parvum. I. Potentiated tumor-specific immunity and its therapeutic limitations.

The anti-tumor mechanism in mice induced by a subcutaneous injection of syngeneic tumor cells admixed with Corynebacterium parvum was investigated. When mice were implanted in a hind footpad with x 2 1096) tumor cells admixed with 100 microgram C. parvum, the tumor that emerged grew progressively for about 9 d and then underwent progressive and complete regression. It was found that this C. parvum-induced regression was associated with the acquisition of a systemic, T cell-mediated mechanism of immunity to tumor-specific transplantation antigens, which enabled the host to cause the regression of an untreated test tumor growing simultaneously at a distant site. The generation of a C. parvum-potentiated anti-tumor response was dependent on the presence of tumor cells in close association with C. parvum, tumor immunogenicity, and the quantity of tumor antigen in the admixture. The anti-tumor immunity was specific for the tumor in the therapeutic admixture and could be adoptively transferred to normal recipients with Thy-1.2-positive lymphocytes, but not with serum. Complete regression of a distant test tumor by the C. parvum-tumor admixture was limited to tumors below a certain critical size.

Mechanisms of anti-tumor action of Corynebacterium parvum. II. Potentiated cytolytic T cell response and its tumor-induced suppression.

It was shown that subcutaneous implantation of P815 tumor cells admixed with Corynebacterium parvum resulted in the emergence of a tumor that grew for 9-10 d and then regressed. The onset of tumor aggression was preceded by the substantial generation in the draining lymph node and spleen of T cells capable of specifically lysing P815 target cells in vitro. The finding that the magnitude of this cytolytic response was much greater than the cytolytic response to a control tumor that grew progressively is consistent with the hypothesis that the anti-tumor action of C. parvum is based on its capacity to augment the production of T cells sensitized to tumor-specific transplantation antigens. This adjuvant action of C. parvum was revealed by additional experiments in which irradiated, nonreplicating tumor cells were substituted for living tumor cells in the admixture. The results support the conclusion that the potentiated cytolytic response to subcutaneous injection of an admixture of irradiated tumor cells and C. parvum is responsible for the ability of this admixture to cause the regression of a test tumor growing at a distant site. Finally, it was shown that the failure of the therapeutic admixture to cause the regression of distant test tumors above a certain size was associated with a failure of the admixture to cause a potentiated, anti-tumor cytolytic response. We discussed the possibility that this failure was caused by the presence of a tumor-induced state of immunosuppression.