Automating home care. Technology's impact on home healthcare providers.
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Ambulatory Care Information Systems↗
Biomedical subjects
E Klein
Publications and source records attributed to E Klein.
Purification of recombinant adenomatous polyposis coli polypeptide chains from E. coli extracts by continuous-elution electrophoresis.
A polypeptide chain encoded by the exons 1-3 of the human adenomatous polyposis coli (APC) tumor suppressor gene was expressed as a maltose binding fusion protein (MBP) in E. coli to be used for immunization purposes. It turned out, that the APC-MBP fusion product of 60 kDa was deposited in bacterial inclusion bodies and, in addition, it was not retarded by an amylose affinity column, most probably due to an altered conformation of the chimeric molecule. For this reason, we established an alternative purification scheme, which took advantage of SDS-extraction followed by a high-resolution two-step continuous-elution electrophoresis (CEE) procedure. This purification method allowed us to obtain high yields of pure human APC exon 1-3-encoded proteins. The final yield of the pure APC polypeptide chains was estimated to represent 5-8% of the amount of SDS-extracted E. coli lysate subjected to the first cycle of CEE. The purified APC molecules were successfully used for the development of specific antibodies. The CEE procedure described here represents a general purification method which is valuable in cases where fusion proteins are deposited as inclusion bodies in bacteria, or if affinity chromatography is precluded due to a conformation-induced lack of ligand binding of the chimeric molecule.
Adenomatous Polyposis Coli↗
Evaluation of a peptide-based solution for peritoneal dialysis.
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Absorption↗
Metallothionein gene expression in bladder cancer exposed to cisplatin.
Some 68% of bladder tumors will respond to cisplatin-based chemotherapy but only 30% will have a durable response. Recent studies have suggested that the metallothionein (MT) gene may produce cisplatin drug resistance in cell lines. To determine the role of MT gene overexpression in human tumors resistant to cisplatin, we evaluated 19 bladder tumors, seven of which had been exposed to cisplatin, for MT mRNA expression. By Northern analysis, four of the seven tumors exposed to cisplatin overexpressed the MT gene compared to untreated tumors. Of the three treated tumors without MT overexpression, one was a relapse 4 yr after the last dose of cisplatin and the other two received only one dose of chemotherapy. MT gene overexpression was found in some tumors that had failed cisplatin chemotherapy and may be a mechanism for drug resistance in bladder cancer.
Base Sequence↗
HLA-B5-restricted auto-tumor-specific cytotoxic T cells generated in mixed lymphocyte-tumor-cell culture.
T-cell-enriched lymphocyte populations derived from the malignant exudate of a patient with ovarian carcinoma were exposed to autologous tumor cells in the mixed lymphocyte-tumor-cell culture (MLTC) and propagated for 42 days. Proliferation of lymphocytes depended on exposures to autologous tumor cells and on the presence of IL-2. After 7 days, the MLTC-lymphocytes lysed K562 and the autologous tumor cells. The latter effect was not inhibited by monoclonal antibodies (MAbs) reactive with MHC class-I antigens or with CD3. After 7 restimulations, the culture was enriched in CD8+ cells (92%) and showed selective lytic activity against the autologous tumor. This function was inhibited by the alpha-class I or alpha-CD3 MAbs, and also by antibodies reactive with the HLA B locus or B5 allele products. The antibodies reactive with HLA A molecules had no such effect. It seems therefore that the function of the CTLs was restricted by HLA B5. Analysis of the TCR beta genes indicated clonal T-cell expansion in this culture. This MLTC was 1 of 21 initiated with 11 blood- and 10 tumor-derived lymphocyte (TIL) populations prepared from the malignant effusions of ovarian carcinoma patients. None of these ex-vivo lymphocytes lysed autologous tumor cells. In 17 MLTCs the lymphocytes did not proliferate, and in 3 cultures the proliferation was maintained only for 2-3 weeks. In 3 of 4 cultures auto-tumor cytotoxicity was induced.
Alleles↗
MHC class-I-restricted auto-tumor-specific CD4+CD8- T-cell clones established from autologous mixed lymphocyte-tumor-cell culture (MLTC).
Autologous mixed lymphocyte-tumor cell cultures (MLTC) were initiated with cytokine (IFN gamma and TNF alpha)-treated ex-vivo tumor cells of lung, ovarian, breast and stomach carcinomas. The cytokine-treated tumors expressed class-I but not class-II molecules. Although the proportion of CD8+ lymphocytes increased in the bulk culture of MLTCs, in 5/7 experiments the majority of the established T-cell clones were CD4+. Among the CD8+ clones a high proportion (77%) was cytotoxic, while the proliferative response was more frequent among the CD4+ clones (70%). In 4/26 cytotoxic T-lymphocyte (CTL) clones (3/17 CD4+ and 1/9 CD8+), derived from a patient with class I+ class II- stomach carcinoma, lysis was restricted to the autologous tumor cells. These auto-tumor-specific clones did not lyse the autologous ConA blasts, the 5 allogeneic ex-vivo tumors, the NK-sensitive K562 or the relatively sensitive Daudi cells. The cytotoxicity of these clones was inhibited by pre-incubation of the tumor cells with W6/32 (alpha-class I) MAb, or by preincubation of the lymphocytes with OKT3 (alpha-CD3) MAb. The alpha-CD4 (OKT4) MAb had only a marginal effect on the CD4+ clones, while the lytic function of the CD8+ clone was inhibited by the alpha-CD8 (OKT8) MAb. The 3 CD4+ CTL clones also responded with proliferation to the autologous tumor cells. This proliferative response was inhibited by the presence of W6/32 MAb. Our results indicate that the auto-tumor lysis exerted by CD4+ CTL clones was restricted by the class-I antigens, and that the CD4 molecules of the clones were not essential for the lytic interaction.
Antibodies, Monoclonal↗
Effect of TGF-beta 1 on the EBV-induced transformation of human lymphocyte cultures.
We tested the effect of TGF-beta 1 on the EBV-induced activation and immortalization of human B lymphocytes. In lymphocyte cultures of EBV-seropositive individuals, T cells can inhibit the EBV-induced transformation of B cells. We found that in the presence of TGF-beta 1 the transformation was more efficient, due to the inhibition of this function. TGF-beta 1 inhibited the EBV-induced proliferation of purified B-cell cultures. Its effect was strongest when added at the beginning of the culture. Added later, the inhibition gradually decreased and was lost by the 4th day. After 10 to 14 days, in the cultures initiated with TGF-beta 1, the number of cells was higher and they formed larger aggregates as compared with control cultures. Thus, TGF-beta 1 modifies EBV-induced transformation in a complex way. It inhibits the activation of B cells but does not affect those already activated. Once they acquire the immortalized state, the B cells are even stimulated by TGF-beta 1.
B-Lymphocytes↗
Increased motor activity and recurrent manic episodes: predictors of rapid relapse in remitted bipolar disorder patients after lithium discontinuation.
Ten patients with remitted bipolar illness on lithium maintenance therapy underwent placebo-controlled lithium discontinuation. Clinical ratings and recording of sleep-wake activity using wrist-worn actigraphs were carried out before and after lithium discontinuation. Seven patients experienced relapse into mania or hypomania within the first 3 months after lithium discontinuation. Actigraphic recordings revealed that patients who relapsed had higher baseline levels of daytime motor activity than patients without relapse. This may suggest that motor activity can be a sensitive marker of subclinical manic tendencies and early relapse following lithium discontinuation.
Adult↗
Expression of the complement regulatory proteins CD21, CD55 and CD59 on Burkitt lymphoma lines: their role in sensitivity to human serum-mediated lysis.
On a panel of nine human B cell lines we showed that the expression of the complement regulatory factors complement receptor type 2 (CR2; CD21), decay-accelerating factor, (DAF; CD55) and homologous restriction factor (HRF20, CD59) is not correlated. All lines expressed DAF, six lines carried detectable amounts of CR2 and three carried HRF20. Upon incubation in human serum, under conditions which allowed the activation of complement through the alternative pathway, the CR2-carrying lines bound C3 fragments and two of them (Ramos and one of its two sublines) were damaged. These two lines had the lowest DAF expression, less than 50% of the cells reacted with the IA10 monoclonal antibody. By modulating the expression of the complement regulatory molecules, the lytic sensitivity of the B cell lines could be altered. Blockade of DAF on the HRF20-, CR2+ lines with the specific monoclonal antibodies increased their sensitivity to lysis by human serum. With the DAF- and HRF20+ cells significant lytic effect was obtained only when they were pretreated with both of the specific antibodies. Interferon-gamma or tumor necrosis factor-alpha treatment elevated the amount of CR2 on the low-CR2 expressor line (Ramos/HR1K) which thereafter bound higher amounts of C3 fragments and was lysed when incubated in human serum. This line had relatively low DAF level and lacked HRF20. The cytokine treatment did not alter the expression of these molecules. The CR2+ Ramos and the CR2- Rael cells were treated with 5-azacytidine which induced HRF20 and increased DAF expression. In parallel with this change Ramos cells became resistant to C-mediated lysis. The experiments with the panel of human B cell lines showed thus that cytolysis through activation of complement in homologous serum can be regulated at several steps by cell surface molecules. While expression of CR2 was required for C3 fixation, DAF and HRF20 inhibited lysis. By independent modulation of the quantities of these molecules, cells acquired or lost their sensitivity.
Antibodies, Monoclonal↗
A trypsin-like serine protease activity on activated human B cells and various B cell lines.
We have studied the trypsin-like serine protease activity of human tonsillar B lymphocytes. The lysate of the low-density, in vivo activated B cells as well as the lysate of cells stimulated with anti-human IgM F(ab')2 show elevated trypsin-like serine protease activity compared to the resting subset as monitored by the cleavage of Tos-Gly-Pro-Arg-pNA. The cleavage is sensitive to N-tosyl-L-lysyl-chloromethyl ketone and benzamidine but not to iodoacetamide. Experiments with intact cells give similar results. The finding that the intact cells hydrolyze the substrate, while their supernatant does not, suggests that the protease activity is cell membrane associated. It is possible that C3 is a substrate of the enzyme since the activated B cells cleave C3, whereas the resting B cells do not, and also C3 inhibits the enzyme-substrate reaction. In addition to the ex vivo B cells, we studied the serine protease activity of certain well-characterized B cell lines. The results show a correlation between the phenotype and the enzyme expression of the cell lines. BL41, an Epstein-Barr virus (EBV)-negative Burkitt lymphoma line, with a resting phenotype, has low activity, while its EBV genome-carrying convertants E95-A-BL41, E95-C-BL41, EHR-A-BL41 and BL41/95 that have the phenotype of activated B cells, have high proteolytic activity. The lymphoblastoid cell line WW-1-LCL which has the phenotype of an immunoblast, has the highest serine protease activity. On the basis of the above data, we suggest that a rather tight correlation exists between the degree of activation and the appearance of serine protease(s) on the surface of human B cells.
B-Lymphocytes↗
Peripheral noradrenergic function during chronic lithium treatment in the rat.
Noradrenergic function was evaluated in the rat vas deferens following chronic treatment with lithium (30 mM in the diet for 2 weeks followed by 50 mM for 1 week). No alteration could be detected in: a) 3H-noradrenaline release evoked by electrical field stimulation, b) alpha 1-adrenoceptor function as assessed by contractile response to noradrenaline, c) active neuronal uptake of 3H-noradrenaline, d) alpha 2-adrenoceptor activity, as assessed by yohimbine-enhancement of evoked release of 3H-noradrenaline. The data argue against a primary action of lithium on the noradrenergic nerve. On the other hand, these findings do not rule out the possibility that the function of CNS noradrenergic neurons could be altered as a result of the known effects of lithium on the phosphatidyl-inositol system, and the greater sensitivity of CNS neurons to depletion of the neuronal membrane inositol pool.
Animals↗
The polysaccharide K (PSK) potentiates in vitro activation of the cytotoxic function in human blood lymphocytes by autologous tumour cells.
We have studied the effect of polysaccharide K (PSK) in the in vitro recognition of ex vivo carcinoma, sarcoma and lymphoma cells by the autologous blood lymphocytes. In 4/25 experiments PSK treatment activated the lymphocytes for auto-tumour lysis. Tumour cells alone generated lytic activity both in short- (16 h) and in long-term (6 days) mixed lymphocyte/tumour cell cultures (MLTC), in 2/12 and 3/13 cases respectively. The tumours that activated the lymphocytes expressed high levels of major histocompatibility complex (MHC) class I molecules. In vitro cytokine (interferon gamma and tumour necrosis factor alpha) treatment of the tumour cells elevated the amounts of class I antigens and the treated cells acquired stimulatory potential. When PSK was added to the MLTC, in which untreated tumour cells were used, lytic potential was induced in 9/13 short-term and in 11/12 long-term cultures. It is noteworthy that in the presence of PSK the untreated, negative or low-class-I-expressor tumours also activated the cytotoxic function of the lymphocytes in 4/5 long-term and in 6/7 short-term cultures. Even in the case of those lymphocytes that could be activated by PSK or tumour cells alone, the simultaneous exposure was more efficient. The effect of PSK was dose-dependent, being optimal at 1 micrograms/ml and 10 micrograms/ml. The presence of EDTA and/or cytochalasin B in the cytotoxic test performed with the activated effectors abrogated the lysis, indicating the requirement of contacts with the effector cells.
Cells, Cultured↗
Assembly of MHC class I molecules in ex vivo carcinoma cells induced by IFN-gamma or by a binding peptide.
It has been reported that the assembly of MHC class I molecules in mutagenized cell lines could be induced by specific binding peptides. We have now demonstrated that the defect in assembly between heavy and light chains of class I molecules naturally occurred in tumor cells of one spontaneous ovarian carcinoma detected by one-dimensional isoelectric focusing of immunoprecipitates with anti-monomorphic class I MAb (W6/32) and by immunostaining with free heavy chain and beta 2m-specific MAbs. In vitro treatment of the tumor cells with IFN-gamma induced the assembly and surface expression of majority class I molecules (A2.1, B7, B15, Cw6, Cw7 out of A2.1, A2*, B7, B15, Cw6, Cw7). Moreover, assembly of A2 and Cw6 was induced by exposure of the tumor cells to a HLA A2-binding peptide K62 derived from influenza A matrix protein. Autologous blood T lymphocytes were activated in mixed lymphocyte-tumor cell culture (MLTC) by the IFN-gamma-treated but not by the unmanipulated tumor cells. Although activated lymphocytes damaged both IFN-gamma-treated and untreated tumor cells, the alpha class I MAb (W6/32) efficiently inhibited the lysis of IFN-gamma-treated targets, but not the untreated targets. These results indicate that the defect of MHC class I assembly may result in the escape of tumor cells from immune response.
Amino Acid Sequence↗
Generation of cytotoxicity with various MHC class I restrictions against autologous LCL by stimulating the lymphocytes with autologous and/or allogeneic LCLs sharing HLA alleles with the responder.
We exposed human blood lymphocytes to autologous and to allogeneic lymphoblastoid lines (LCLs), each alone or in combination, and analyzed the MHC Class I restriction pattern of the generated auto-LCL reactive cytotoxicity. In the cultures of two EBV-seropositive, HLA A11-positive individuals the majority of cytotoxic lymphocytes generated after repeated stimulation with autologous LCL were restricted by this molecule. One of the cultures was subjected to various stimulation strategies. A relatively low proportion of HLA A2- and HLA B7-restricted cytotoxic T cells could be detected in the autostimulated cultures. Such cells were enriched at the expense of A11-restricted ones by stimulating with allogeneic LCLs which lacked HLA A11 but expressed A2 or B7. Interestingly, stimulation of the lymphocytes with only allogeneic LCL also generated autoreactive CTLs. Thus, by including or using exclusively allogeneic LCL stimulators, the CTL fractions represented by few cells could be enriched.
Alleles↗
Cancer immunotherapy: are the results discouraging? Can they be improved?
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Animals↗
Characterization of a human renal cell carcinoma specific cytotoxic CD8+ T cell line.
Human renal cell carcinoma (RCC) is one of the tumors most sensitive to immunotherapy and in that regard it is similar to malignant melanoma. Clinical studies reporting responses to therapy suggested that a host immune response may be involved in the antitumor activity induced by immunotherapy in these tumors. Although detection of a specific T cell response to melanoma has been well documented, this has not been the case for RCC. The lytic response of interleukin-2 (IL-2) cultured tumor-infiltrating lymphocytes (TILs) from RCC has been nonspecific. However, in this report we describe a CD8+ TIL line derived from a primary RCC tumor that displays specificity for the autologous tumor. This line is lytic for autologous RCC but does not lyse autologous lymphoblasts, allogeneic RCC, or tumor cell lines of other histologic types. It also proliferates specifically to the autologous tumor in the absence of exogenous IL-2. However, the addition of low dose IL-2 to the cultures can significantly augment its proliferative response. When stimulated with autologous RCC but not allogeneic RCC the CD8+ line will produce interferon-gamma (IFN-gamma). It appears that recognition of RCC by this TIL line is through the TCR/CD3 complex because anti-CD3 antibody blocks the lytic activity, proliferation and IFN-gamma production of the line in response to the autologous tumor. Additional studies illustrate that cytotoxic T lymphocytes with apparent specificity for the autologous tumor are present in unseparated cultured TILs that can be detected by clonal analysis. Collectively these results suggest that there is a specific T cell response to human RCC.
CD8 Antigens↗
The effects of a social support group on depression, maternal attitudes and behavior in new mothers.
The present study examined the effects of a postpartum Social Support Group intervention on mood state, attitudes and behavior of new mothers. Intervention conditions consisted of an eight session Social Support intervention (n = 44), a no intervention condition (n = 83) and a Group-by-Mail intervention (n = 15). Attitudinal and behavioral assessments were made before and after the interventions at 6 and 20 weeks postpartum, respectively. The primary results indicate that regardless of intervention condition mothers undergo an improvement in mood from 2 weeks to 5 months postpartum. Although the Social Support intervention did not alleviate maternal depression and, in fact, may be detrimental to depressed mothers' self-confidence, it did increase mothers' proximal attention to their infants.
Adult↗