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Biomedical subjects

R K Puri

Publications and source records attributed to R K Puri.

At least 91 records · Page 5Linked to original sources
PubMed1995

Human glioma cells overexpress receptors for interleukin 13 and are extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin.

Recently, we have demonstrated that a spectrum of human adenocarcinoma cell lines express binding sites for interleukin 13 (IL-13). These cells are killed by a chimeric protein composed of human (h) IL-13 and a derivative of Pseudomonas exotoxin, PE38QQR (Debinski et al., J. Biol. Chem., 270: 16775-16780, 1995). The cell killing was hIL-13- and hIL-4-specific, indicating that a common binding site for the two cytokines is present in several solid tumor cell lines. Herein, we report that an array of established glioma cell lines is killed by very low concentrations of hIL-13-PE38QQR, often reaching <1 ng/ml (<20 pM). Glioma cells express up to 30,000 molecules of IL-13 receptor/cell which has intermediate affinity toward hIL-13. hIL-13-PE38QQR is more active (up to 3 logs difference in cytotoxic activities) than are the corresponding chimeric toxins containing hIL-4 or hIL-6. The cytotoxic action of hIL-13-PE38QQR is blocked by an excess of hIL-13 on all cell lines studied, and it is not neutralized by hIL-4 on some of these cells. Our results show that human brain cancers richly express receptors for IL-13. Furthermore, the interaction detected previously between receptors for IL-13 and IL-4 on solid tumors cell lines is of a qualitatively different character in U-251 MG and U-373 MG glioma cells. The receptor for IL-13 may represent a new marker of brain cancers and an attractive target for anticancer therapies.

ADP Ribose Transferases↗
PubMed1994

Site-specific conjugation to interleukin 4 containing mutated cysteine residues produces interleukin 4-toxin conjugates with improved binding and activity.

Fusion of a ligand to another protein frequently impairs the binding of the ligand. Recombinant toxins composed of mutants of Pseudomonas exotoxin (PE) fused to the C-terminus of human interleukin 4 (IL4) are cytotoxic to IL4 receptor- (IL4R-) bearing tumor cells but bind to the IL4R with only 1% the affinity of IL4. We have developed a method to connect a toxin to a ligand which allows the junction to be moved to a location on the ligand which would minimize the binding impairment. We designed mutants of IL4 in which residue 28, 38, 68, 70, 97, or 105 was substituted with cysteine. All purified mutants bound to the IL4R with 60-100% the affinity of IL4, indicating that the IL4 structure was essentially unchanged. The IL4 mutants were then each conjugated through a disulfide bond to PE35, a truncated form of PE which contains a single cysteine. IL4 conjugated to PE35 at residue 28, 38, or 105 of IL4 bound with 10-fold improved affinity and was 10-fold more cytotoxic than the recombinant IL4-toxin in which PE is fused to position 129 at the C-terminus of IL4. IL4 containing PE35 conjugated at position 68, 70, or 97 had lower binding affinity and cytotoxic activity. These results indicate that the location of the ligand-protein junction can be selectively moved to enhance conjugate effectiveness, and implications could be made regarding which regions of IL4 are important for binding.

ADP Ribose Transferases↗
PubMed1994

Interleukin-4 receptors expressed on tumor cells may serve as a target for anticancer therapy using chimeric Pseudomonas exotoxin.

Interleukin-4 receptors (IL4R) are present on a wide variety of human cancer cells derived from both hematopoietic and epithelial malignancies. We have targeted IL4R on a human solid tumor xenograft with chimeric proteins composed of human IL4 (hIL4) and 2 different mutant forms of a powerful bacterial toxin, Pseudomonas exotoxin A (PE). The 2 chimeric toxins, termed hIL4-PE4E and hIL4-PE38QQR, showed specific, hIL4R-dependent and dose-dependent antitumor activities. Neither of the chimeric toxins showed antitumor potency when the ADP-ribosylation activity of the toxin was inactivated by mutagenesis. One of the chimeras, hIL4-PE38QQR, caused a complete although transient regression of established solid tumors. These observations indicate that hIL4-PE chimeric proteins should be further evaluated for the treatment of human malignancies.

ADP Ribose Transferases↗
PubMed1994

Human neurological cancer cells express interleukin-4 (IL-4) receptors which are targets for the toxic effects of IL4-Pseudomonas exotoxin chimeric protein.

Glioblastoma, glioma or neuroblastoma cells were examined for the expression of IL-4 receptors (IL-4R) by flow cytometric analysis and 125I-IL-4 binding. These cancer cell lines expressed IL-4R which were of high affinity (KD = 700 x 10(-12) M) on glioblastoma cells. To investigate the function of these receptors and to target potent cytotoxic antitumor agents to human neurological cancers, we utilized IL4-PE4E, which is composed of IL-4 and mutant Pseudomonas exotoxin (IL4-PE4E). This chimeric molecule was cytotoxic toward human glioblastoma, neuroblastoma and glioma tumor cells in a dose-dependent manner. The cytotoxicity of IL4-PE4E was specific, since it was neutralized by excess IL-4, and by an anti-IL-4 monoclonal antibody in all types of brain tumor tested. IL2-PE4E and IL6-PE4E were not cytotoxic, nor was an IL4-PE4E mutant lacking ADP-ribosylating activity, indicating the IL4-PE4E-mediated cytotoxicity of the brain tumor cells required both IL-4R binding and enzymatic toxin activity. These data indicate that human neurological cancer cells express IL-4R which are targets for the cytotoxic effects of IL4-toxin. In addition, our data also suggest that IL4-PE4E should be studied further as a potential treatment for human neurological cancers.

ADP Ribose Transferases↗
PubMed1994

A circularly permuted recombinant interleukin 4 toxin with increased activity.

Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor. With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the ligand. However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor. To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fragment encoding circularly permuted interleukin 4 (IL4), termed IL4(38-37). This was accomplished by placing a start codon before position 38, connecting codons 1 and 129 with a sequence encoding a peptide linker, and placing a stop codon after codon 37 of IL4. IL4(38-37) was fused via its new carboxyl terminus, Lys37, to a truncated form of Pseudomonas exotoxin. The purified circularly permuted IL4-toxin bound to the IL4 receptor with 10-fold higher affinity than an IL4-toxin in which the toxin was fused to the carboxyl terminus of IL4. Circular permuteins of growth factors can improve the effectiveness of recombinant fusion proteins, because the junction can be moved to a site on the growth factor which allows it to bind with higher affinity.

ADP Ribose Transferases↗
PubMed1994

IL-10 inhibits HIV-1 replication and is induced by tat.

Interleukin 10 (IL-10) is produced by TH2 lymphocytes and regulates both lymphoid and myeloid cells. In the present study we demonstrate that IL-10 is expressed and produced spontaneously in the peripheral blood mononuclear cells (PBMCs) of all HIV-1 infected individuals tested, 3 of 19 cases of HIV-negative lymphoma and none of five healthy controls. IL-10 mRNA was detectable in both monocytes/macrophages and T lymphocytes isolated from PBMCs of HIV infected patients. We have also shown that infection of promonocytic (U937) and T (H9) cell lines with HIV stimulates IL-10 secretion. Furthermore, a T cell line (H9) stably transfected with a HIV tat expression-vector secreted higher levels of IL-10. We have also demonstrated that rhIL-10 inhibited HIV-1 replication in infected monocytes and PBMCs in a dose dependent manner. IL-10 may thus participate in long latency between HIV-1 infection and development of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Human renal cell carcinoma cells are sensitive to the cytotoxic effect of a chimeric protein composed of human interleukin-4 and Pseudomonas exotoxin.

We have previously demonstrated that functional high-affinity interleukin-4 receptors (IL-4R) are expressed on human renal cell carcinoma (RCC) cells (N. I. Obiri et al., J. Clin. Invest. 91, 88, 1993). In the present study, we examined the cytotoxic effect (determined by inhibition of protein synthesis) of a chimeric protein composed of human IL-4 and Pseudomonas exotoxin (PE) on human RCC tumor samples obtained from patients undergoing nephrectomy. The chimeric gene encoding hIL4-PE4E was constructed by fusing a cDNA clone for human IL-4 to the 5' end of a mutated cDNA encoding a full-length PE molecule. This gene was expressed in Escherichia coli, and large quantities of this recombinant protein were isolated to more than 95% purity. This chimeric protein, hIL4-PE4E, was highly cytotoxic to all six RCC cell lines examined. The concentration of hIL4-PE4E at which 50% inhibition of protein synthesis was obtained ranged from < 1 ng/ml (12 pM) to 10 ng/ml (120 pM) in five of the six isolates of RCC and 40-70 ng/ml in one other. A mutant chimeric protein which can bind to IL-4R but lacks the ADP ribosylation activity of PE was not cytotoxic to the RCC cells. The cytotoxic effect of hIL4-PE4E was IL-4R mediated because a fourfold molar excess of IL-4 abrogated the cytotoxic effect of hIL4-PE4E. A neutralizing monoclonal antibody to IL-4 also abrogated the cytotoxic effect of hIL4-PE4E. hIL4-PE4E showed very little cytotoxic activity to a normal human umbilical vein endothelial cell line (ID50 = 1000 ng/ml) and a human fibroblast cell line (ID50 approximately 400 ng/ml). Nonactivated human peripheral blood lymphocytes (PBL) were also insensitive to hIL4-PE4E (ID50, approximately 500 ng/ml), whereas phytohemagglutinin-activated PBL were highly susceptible to the cytotoxic effect of hIL4-PE4E (ID50, approximately 4 ng/ml). These data indicate that hIL4-PE4E may be a useful agent for the treatment of human RCC without affecting normal and resting immune cells.

ADP Ribose Transferases↗
PubMed1994

A chimeric protein comprised of IL-4 and Pseudomonas exotoxin is cytotoxic for activated human lymphocytes.

IL4-Pseudomonas exotoxin (IL4-PE4E) is a chimeric molecule in which human IL-4 is genetically fused to the mutated binding domain of Pseudomonas exotoxin. This molecule binds specifically to human IL-4 receptor-bearing cells. IL4-PE4E was extremely cytotoxic to highly purified anti-CD3-activated CD8+ T lymphocytes. The cytotoxic activity of this molecule was dependent on the activation state of CD8+ T cells: 3- and 4-day activated T cells were very susceptible to the cytotoxic activity of IL4-PE4E compared with 0- to 2-day activated cells. PHA-activated lymphocytes and PBL activated in mixed lymphocyte reaction were also highly sensitive to IL4-PE4E. CD16+ and/or CD56+ highly purified NK cells or highly purified, IL-2-activated NK cells were also very sensitive to the cytotoxic effect of IL4-PE4E. IL-2-activated LAK cells had little susceptibility after 1 day but were very sensitive to IL4-PE4E after 3 days. The cytotoxic effects of IL4-PE4E were mediated through a ligand receptor interaction because excess rIL-4 abrogated these effects as did a neutralizing Ab to human IL-4. A chimeric mutant protein that can bind to IL-4 receptors but lacks the ability to inhibit protein synthesis was not cytotoxic to activated lymphocytes. The IL4-PE4E-mediated cytotoxicity of activated T cells correlated with the level of expression of IL-4 receptors on these cells. CD8+ T cells activated for 3 days expressed the highest density of IL-4 receptors compared with 1- or 2-day activated cells. Among two chimeric toxins tested only IL4-PE4E was cytotoxic to 2-day anti-CD3-activated CD8+ T lymphocytes, whereas IL6-PE4E was not active at all. These studies suggest that human IL4 toxin could be a potent agent for the elimination of activated lymphocytes in allograft rejection, some autoimmune diseases, or treatment of lymphomas and leukemias.

ADP Ribose Transferases↗
PubMed1994

Regulatory review of cellular and gene therapies: an overview of the process.

Cell and gene therapies, using several different approaches, have been proposed for a variety of genetic diseases, cancer and AIDS. The major regulatory review process in the US consists of an institutional review board, the recombinant DNA advisory committee (RAC) and the Food and Drug Administration (FDA). Within the Center for Biologics Evaluation and Research, the Division of Cellular and Gene Therapies has been formed to primarily review investigational new drug applications (INDs) for cellular and gene therapies. Several appropriate "points to consider" documents have been prepared and the RAC has approved over 40 clinical protocols. Advances in biotechnology and the scientific basis for these advances are changing rapidly. Although a flexible, case-by-case approach is necessitated by these rapid changes, regulatory concerns common to all biologicals administered to human subjects remain unchanged. These include safety, efficacy, purity, potency, quality control and assessment, and reproducibility of individual lots. The goal of the review process is a prompt, complete and meticulous review. The emphasis of a pre-IND meeting is toward a working relationship between the sponsor and the FDA prior to the phase I, II and III clinical trials. A timely and ongoing evaluation of pre-clinical testing cannot be overemphasized in this rapidly growing and changing field. The development of a working relationship at this stage will ensure a seamless integration of the IND process with the product and establishment license applications. Because replication-competent retrovirus (RCR) represents a potential for pathogenicity, the FDA is recommending a conservative approach to RCR testing.

Animals↗
PubMed1994

1H- and 13C-nmr assignments and structural determination of a novel glycoalkaloid from Solanum platanifolium.

Four steroidal glycoalkaloids, solasonine [2], solamargine [3], khasianine [4], and ravifoline [5], with solasodine [1] as the aglycone moiety, have been isolated from Solanum platanifolium. The present study involved the characterization of the novel compound 5 by one- and two-dimensional nmr techniques and has also led to the unambiguous and total 13C- and 1H-nmr assignments of all of these steroidal glycoalkaloids and the direct elucidation of their glycosidic linkages.

Carbohydrate Sequence↗
PubMed1994

Expression of high-affinity IL-4 receptors on human melanoma, ovarian and breast carcinoma cells.

It has previously been shown that murine sarcoma cells express high-affinity IL-4 receptors (IL-4R) which are internalized after binding to the ligand (Puri et al., Cancer Res 1991; 51:3011-7). We have also reported that human renal cell carcinoma cells express high-affinity IL-4R, and IL-4 inhibits tumour growth in vitro (Obiri et al., J Clin Invest 1993; 91:88). In this study we investigated the expression and function of IL-4R on other human solid tumours. Human melanoma, ovarian carcinoma and breast carcinoma cell lines were assessed for the cell surface expression of IL-4R by radio-ligand receptor binding and for IL-4R gene expression by Northern blot analysis. Primary cultures of mesothelioma and neurofibrosarcoma cells were similarly investigated. Human melanoma, ovarian carcinoma and breast carcinoma cell lines expressed IL-4R on their cell surface with a dissociation constant (Kd) of 140-549 pM. These tumour lines expressed a single 4 kb species of mRNA for IL-4R. Similarly, primary cultures of mesothelioma and neurofibrosarcoma cells were positive for the IL-4R mRNA by Northern blot analysis. Fresh, non-cultured mesothelioma and neurofibrosarcoma tumour sections were also positive for the presence of IL-4R as determined by immunohistochemistry of frozen sections using anti-IL-4R antibody. In order to study possible functions of IL-4R, we evaluated the effects of IL-4 on cell growth and its effect on MHC antigen expression in the presence or absence of interferon-gamma (IFN-gamma). In tissue culture, IL-4 reduced the growth of tumour cell lines and primary cell cultures studied. IL-4 had very little effect on MHC class I antigen expression on ovarian, breast and melanoma cell lines; however, MHC class II (HLA-DR) expression was enhanced on melanoma and breast carcinoma cells. IL-4 also enhanced the IFN-gamma-induced class II expression on melanoma and breast carcinoma cells. Taken together, our observations indicate that IL-4R are expressed on a variety of human solid tumours and these receptors may be functional. IL-4 alone and in combination with IFN-gamma may play a role in host immune response against cancers.

Breast Neoplasms↗
PubMed1994

Growth and major histocompatibility antigen expression regulation by IL-4, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on human renal cell carcinoma.

We have recently shown that human renal cell carcinoma (RCC) tumour lines express high-affinity IL-4 receptors. Binding of IL-4 to RCC cells induced a growth inhibition in the range of 20-68%. To enhance the growth inhibitory effect of IL-4, we have tested the effects of two additional cytokines capable of directly affecting tumour cell growth. IFN-gamma caused a significant inhibition of RCC tumour cell growth (up to 70%) in a dose-dependent manner, whereas the effect of TNF-alpha was more limited (0-20% inhibition). The addition of IL-4 to IFN-gamma on RCC cells sensitive to IL-4 induced a greater inhibition of cell growth than that seen with each cytokine alone. IL-4 and IFN-gamma rendered RCC cells more responsive to the inhibitory effect mediated by TNF-alpha. The combination of TNF-alpha with IL-4 and IFN-gamma induced an optimal growth inhibition (up to 90-98%) of RCC cells. In addition to a direct anti-proliferative effect, we have demonstrated that these cytokines can also enhance the expression of MHC antigens on the surface of RCC tumour cell lines which may render the cells more immunogenic. All RCC lines tested expressed class I antigens, but not class II antigens. IFN-gamma induced class II expression and up-regulated the expression of class I antigens on RCC cells. Class II antigen expression was detectable following 48 h incubation, and greater after 72 h with IFN-gamma. IL-4 minimally affected class I expression, whereas TNF-alpha up-regulated class I antigen expression. IL-4 or TNF-alpha did not induce class II expression. The combination of the three cytokines slightly augmented the up-regulation of class I and class II antigens observed with IFN-gamma alone. These observations confirm the direct interaction of IL-4, IFN-gamma and TNF-alpha with RCC tumour cells, both at the level of growth regulation and MHC antigen expression, and suggest a therapeutic potential of the combination of the three cytokines for renal cell carcinoma.

Carcinoma, Renal Cell↗
PubMed1994

Microvascular responses to body tilt in cutaneous maximus muscle of conscious rats.

We investigated microvascular responses to head-up tilt (HUT) and head-down tilt (HDT) in striated muscle of conscious male rats (n = 15; body wt 163 +/- 5 g). To observe the microcirculation in the cutaneous maximus muscle, a transparent polycarbonate chamber (1.5 cm diam) was implanted aseptically into a skin fold created between the shoulders. Rats were trained to sit quietly during HUT and HDT while positioned on a horizontal microscope that rotated in the sagittal plane. At 4-5 days after surgery, arteriole and venule diameters were recorded using videomicroscopy while the rat experienced 10 min each (in random order) of HUT or HDT at 20 degrees or 40 degrees separated by 2-h rest periods. HUT had no affect on microvessel diameter; 20 degrees HDT had little affect. In response to 40 degrees HDT, "large" arterioles (88 +/- 18 microns; n = 10) constricted by 18 +/- 2% (P < 0.05) and "small" arterioles (40 +/- 17 microns; n = 7) dilated by 21 +/- 3% (P < 0.05); this difference suggests variation in mechanisms controlling arteriolar responses. Venules (45 +/- 22 microns; n = 9) exhibited a larger fluctuation in diameter (amplitude 13 +/- 5 microns) during 40 degrees HDT compared with other body positions (amplitude typically 2-3 microns), suggesting that venomotor activity may be induced with sufficient fluid shift or change in central venous pressure. These observations illustrate a viable model for studying microvascular responses to gravitational stress in conscious rats.

Animals↗
PubMed1994

Characterization of interleukin-4 receptors expressed on human renal cell carcinoma cells.

We have recently reported that a variety of solid human tumor cells express high-affinity interleukin-4 receptors (IL-4R). In this study, we have compared structural characteristics of IL-4R expressed on human renal cell carcinoma cells (RCC-WS) and the lymphoid cell lines RAJI (B-cell line) and H9 (T-cell line). In crosslinking studies, the three cell types expressed a predominant 140 kDa IL-4R band. In addition, a 70 kDa band was expressed strongly in H9 cells but only faintly on RCC-WS and RAJI cells. These different species of IL-4R were not observed when crosslinking studies were performed in the presence of excess interleukin-4 (IL-4), indicating IL-4 specificity. A polyclonal anti-IL-4R antibody immunoprecipitated the two species (140 and 70 kDa) in H9 and predominantly the 140 kDa species in RCC-WS tumor cells and RAJI cells. A faint band for the 70 kDa protein was also observed. The affinity of IL-4 binding to its receptor in RCC-WS cells was similar to the binding affinity observed in H9 and RAJI cells examined. However, the RCC tumor cells and B lymphoid cells internalized IL-4R more rapidly compared to T lymphoid cells. Although IL-4R synthesis was similarly inhibited by cycloheximide in all three cell lines, IL-4R expression was more sensitive to actinomycin D inhibition on the RCC-WS and RAJI cells than on H9 cells. Our results suggest that IL-4R expressed on RCC-WS tumor cells are structurally different from those expressed on lymphoid cells because the proportions of IL-4R subunits differ in these cells. Further studies should be performed to determine the identity and functional significance of IL-4R proteins expressed on RCC and immune cells.

Carcinoma, Renal Cell↗
PubMed1994

Simple predictors to differentiate acute asthma from ARI in children: implications for refining case management in the ARI Control Programme.

There is a considerable overlap in the clinical presentation of acute asthma and ARI. According to the current ARI Control Programme recommendations, a child with cough and rapid breathing is overtreated for ARI (pneumonia) with antibiotics and undertreated for asthma with bronchodilators. The present study, therefore, evaluated simple predictors to differentiate these two conditions to refine the recommended case management. In a case control comparison, children between 6 to 60 months age who presented with cough and rapid breathing due to acute asthma (n = 100) and ARI (n = 100) were evaluated. Only 34% of asthmatics had an audible wheeze. Significant independent predictors on multiple logistic regression analysis were number of earlier similar attacks and fever (or temperature). The best predictor for asthma was two or more earlier similar episodes (sensitivity 84%, specificity 84%) followed by temperature < 37.6 degrees C (sensitivity 73% and specificity 84%). Absence of fever, audible wheeze and a family history of asthma had excellent specificities (98-100%) but low sensitivities (20-34%). It is concluded that simple clinical predictors can differentiate acute asthma and ARI. The recommended case management can, therefore, be refined by either: (i) Prescribing bronchodilators and no antibiotics with two or more earlier similar episodes of cough and rapid breathing; or (ii) To further minimize undertreatment for pneumonia, prescribing bronchodilators as above, but denying antibiotics in such cases only if there is audible wheeze or family history of asthma or no fever.

Acute Disease↗