Genetically engineered superantigens in experimental tumor therapy.
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Animals↗
Biomedical subjects
L Ohlsson
Publications and source records attributed to L Ohlsson.
Antibody-targeted superantigens in cancer immunotherapy.
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Animals↗
Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo.
Recruitment of antigen-specific tumor-infiltrating lymphocytes (TILs) is a major goal for immunotherapy of malignant tumours. We now describe that T-cell-activating superantigens targeted to a tumor by monoclonal antibodies induced large numbers of pseudospecific TILs and eradication of micrometastases. As a model for tumor micrometastases, syngeneic B16 melanoma cells transfected with the human colon carcinoma antigen C215 were injected intravenously into C57BL/6 mice and therapy with an anti-C215 Fab fragment-staphylococcal enterotoxin A (C215Fab-SEA) fusion protein reacting with the C215 antigen was initiated when visible lung metastases were established. More than 90% reduction of the number of lung metastases was observed when mice carrying 5-day-old established lung metastases were treated with C215Fab-SEA. The antitumor effect of C215Fab-SEA was shown to be T-cell-dependent since no therapeutic effect was seen in T-cell-deficient nude mice. Depletion of T-cell subsets by injection of monoclonal antibody demonstrated that CD8+ cells were the most prominent effector cells although some contribution from CD4+ cells was also noted. C215Fab-SEA treatment induced massive tumor infiltration of CD4+ and CD8+ T cells, while only scattered T cells were observed in untreated tumors. SEA treatment alone induced a slight general inflammatory response in the lung parenchyme, but no specific accumulation of T cells was seen in the tumor. TILs induced by C215Fab-SEA were mainly CD8+ but a substantial number of CD4+ cells were also present. Immunohistochemical analysis showed strong production of the tumoricidal cytokines tumor necrosis factor alpha and interferon gamma in the tumor. Thus, the C215Fab-SEA fusion protein targets effector T lymphocytes to established tumors in vivo and provokes a strong local antitumor immune response.
Animals↗
Tumor-reactive superantigens suppress tumor growth in humanized SCID mice.
Superantigens are extremely potent activators of T lymphocytes. To develop a tumor-reactive superantigen for cancer therapy, we made a recombinant fusion protein of the superantigen staphylococcal enterotoxin A (SEA) and the Fab region of the C242 monoclonal antibody (C242Fab-SEA), which recognize human colon carcinoma cells. The therapeutic effect of C242Fab-SEA on colon-cancer growth was examined in lymphocyte-engrafted humanized SCID mice bearing intraperitoneally growing Colo205 colon carcinomas. i.v. injections of C242Fab-SEA significantly inhibited tumor growth. The anti-tumor effect required the presence of human T cells in the SCID mice. Optimal therapeutic effects were seen with C242Fab-SEA, but not with C242Fab fragment or SEA alone, demonstrating that both entities of the fusion protein were required. C242Fab-SEA-treated tumors showed a massive infiltration of T cells in the tumor parenchyme, and was accompanied by enhanced expression of ICAM-I and HLA-DR on the tumor cells. The results demonstrate that Fab-SEA fusion proteins convey superantigenicity on tumor cells which evoke T-cell-dependent suppression of tumor growth.
Animals↗
Immunotherapy of human colon cancer by antibody-targeted superantigens.
T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma.
Animals↗
Heterogeneity of renal carcinoma.
Monoclonal antibodies were used to study the expression of three recently characterized basement membrane components and two carbohydrate antigens in 11 renal-cell carcinomas, using immunohistological and biochemical techniques. The expression of several site-specific kidney antigens in renal-cell carcinoma were studied to determine the origin of the carcinoma and if it is possible further classify this type of carcinoma. Tubulointerstitial nephritis antigen (TIN) and two alpha-chains of type IV collagen, alpha 1 (IV) and alpha 3 (IV) were studied. In addition the expression of carbohydrate antigens Lex and SLex, which also exhibit site-specific distribution were characterized. Lex and SLex antibodies stained the majority of the tumours. TIN was expressed in 9 of 11 tumours, the alpha 1 (IV) chain was present in all 11, and the alpha 3 (IV) chain in two of the 11 tumours. Interestingly, the two alpha 3 (IV)-positive tumours were the same two that were negative for TIN. In normal tissue alpha 3 (IV) is found in distal tubules while TIN is found in proximal tubules. Our results are consistent with earlier observations that the proximal tubule is the origin of most renal-cell carcinomas, but the results also indicate that renal-cell carcinoma may originate from the distal tubule.
Antibodies, Monoclonal↗
Immunocytochemical detection of the carbohydrate antigen, Sialyl Lewis(x), in normal human skin and during irritant contact dermatitis.
Sialys Lewis(x) (SLex) is a ligand for the E-selectin and the interaction of E-selectin on the endothelium and SLex on T cells may be important for T-cell migration into the skin. We investigated the expression of SLex on Langerhans cells (LC) in normal skin and on LC repopulating epidermis deprived of LC due to a preceding irritant contact dermatitis. SLex was visualized by fluorescence and light microscopic immunocytochemistry using the monoclonal antibody, CSLEX-1. The results showed that about 40% of LC in normal epidermis express SLex. In the repopulation phase, most of the epidermal cells were CD1a+/SLex. We suggest that SLex is present on epidermal LC that have recently immigrated from the dermis.
Cell Movement↗
Islet cell antibodies, but not glutamic acid decarboxylase antibodies, are decreased by plasmapheresis in patients with newly diagnosed insulin-dependent diabetes mellitus.
The effects of plasmapheresis on islet autoantibody levels, C-peptide (beta-cell function), and hemoglobin-A1c (HbA1c, metabolic control) were tested in a prospective blinded study of 18 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients randomly assigned to receive plasmapheresis (P), carried out as double filtration, or sham (S) treatment at diagnosis and 3 months thereafter. At diagnosis, 6 of 8 patients (75%) in group P and 9 of 10 patients (90%) in group S had islet cell antibodies (ICA), whereas 4 of 8 (50%) and 7 of 10 (70%) patients, respectively, had glutamic acid decarboxylase antibodies (GAD65-Ab), with no significant differences between the groups in ICA and GAD65-Ab levels. After 6 months, P patients showed significantly lower ICA levels than S patients (11 +/- 6 and 128 +/- 47 Juvenile Diabetes Foundation International Units, respectively; P < 0.02) due to an increase in ICA levels in 8 of 9 (88%) of the S patients not seen in P patients (P < 0.002). Concurrently, HbA1c stabilized in P, but not in S, patients and was significantly lower by 24 months (6.58 +/- 0.54% vs. 9.76 +/- 1.21%; P < 0.05). Moreover, fasting C-peptide increased significantly (214 +/- 11 pmol/L; P < 0.05) over the first 6 months in P. After the initial 6 months, ICA levels tended to decrease in all patients and were not detected after 60 months. GAD65-Ab levels were not influenced by plasmapheresis and, also in contrast to ICA, increased significantly (P < 0.05) in the whole study population after 60 months. In fact, 4 initially negative patients became GAD65-Ab positive after diagnosis (in 2 patients > 24 months after diagnosis). We conclude that plasmapheresis of newly diagnosed IDDM patients does not change subsequent GAD65-Ab levels, but ICA are significantly decreased with associated improved C-peptide and HbA1c levels.
Adolescent↗
Acute and long-term biphasic volume alterations in rat type-II somatotrophs during GH secretory stimulation.
The size and shape of growth hormone (GH)-producing rat (type II)-somatotrophs was studied during secretory stimulation by either human GH-releasing hormone (hGHRH(1-29)) or 50 nM extracellular potassium. A new type of perfusion chamber for light microscopy allowed the interpretation of early changes in cell morphology. The exposure to 10 nM hGHRH caused a significant transient decrease in cell volume to 94.4 +/- 2.1% within 2 s. The cell volume recovered to 99.2 +/- 1.3% at 30 s. A second, more gradual volume decrease then followed (60 s), which stabilized at about 92.5 (8 min) and was still present after 20 min of continuous hGHRH exposure. Potassium, 50 mM, gave an immediate and persistent cell volume increase of about 5%. The fluctuation in rat somatotroph volume after hGHRH exposure correlates to a previously observed biphasic GH-secretory pattern involving an initial burst secretion and a second slow phase secretion. The transient early volume decrease may reflect ion fluxes across the membrane and/or the response of the cytoskeleton to calcium mobilization during the GH-secretory onset. The second, persistent, volume decrease closely correlates to the calculated volume of lost GH vesicles.
Animals↗
Cancer pain relief by continuous administration of epidural morphine in a hospital setting and at home.
Fifteen patients with severe pain due to malignancy were treated by continuous epidural morphine infusions. A disposable external pump was used. Patients were treated in a hospital setting or at home for a total of 906 days. Pain intensity was estimated by VAS. The pumps functioned well. Bacterial growth was found in 0.6% of the balloon reservoirs used, while the epidural filters were free from growth. There were no clinical infections. It appears that this delivery system is safe, practical and suitable for use in the home environment.
Aged↗
Effect of N-methyl-D,L-aspartate on isolated rat somatotrophs.
The effect of excitatory amino acid receptor agonists on GH secretion was tested in isolated male rat somatotrophs. N-Methyl-D,L-Aspartate (NMDA) had a dose-dependent stimulatory effect on GH secretion in perifused somatotrophs. The effect was observed already during the first minute after exposure to NMDA and was reversible after its omission. The effect of 1 microM NMDA was inhibited by the NMDA receptor antagonists 10 microM AP7 and 5 microM MK801, and by 5 microM dextromethorphan. L-Glutamate, 100 microM, and 100 microM kainic acid also stimulated GH secretion. The stimulatory effect of NMDA on GH release was paralleled by an increase in 45Ca efflux and an increase in somatotroph intracellular calcium concentration. Efflux of 86Rb (tracer for potassium) was not affected by NMDA. It is concluded that excitatory amino acids can stimulate GH secretion in rats through a direct effect on the somatotrophs.
Amino Acids↗
Morphology and filterability of red blood cells in neonatal and adult rats.
Red blood cell (RBC) deformability in rats of various ages was assessed by filtration (3 micron Nucleopore membranes). Neonatal rat RBC (1 d old) had lower filterability, both in terms of RBC incremental volume (9.97 +/- 1.85 versus 0.33 +/- 0.28 nL at 180 d of age, mean +/- SD, p less than 0.001) and the number of filter clogging particles (25.7 +/- 3.1 versus 18.9 +/- 3.4 RBC x 10(3)/s, p less than 0.001). The lower filterability correlated with a larger RBC volume (169 +/- 12.6 versus 69 +/- 3.2 microns 3, p less than 0.001) and with a wider minimum cylindrical diameter (3.87 +/- 0.15 versus 2.84 +/- 0.05 microns, p less than 0.001). Almost all of the neonatal RBC had a minimum cylindrical diameter exceeding the 3 microns nominal pore size of the filters. The calculated resistance to initial folding was also significantly greater, as indicated by a static bending analysis of initial deformation. However, when the larger size of neonatal RBC was taken into consideration, and thus their greater projected area on which forces are applied, they appear to be at least as deformable as the adult type RBC. This finding may explain the contradiction between RBC filtration experiments and other approaches based on RBC deformations in shear flow, which have been unable to detect a hampered flexibility of neonatal RBC. In view of the more pronounced differences between neonatal and adult RBC in rats than in human subjects, the rat is an interesting model for studying this physiologic phenomenon in newborns.
Aging↗
The correlation between calcium outflow and growth hormone release in perifused rat somatotrophs.
The time-relationships between GH secretion and 45Ca2+ efflux in response to human GRF (hGRF)-(1-29), hGRF-(1-44), 3-isobutyl-1-methylxanthine (IBMX), (Bu)2-cAMP, and high extracellular K+ were studied in perifused cultured rat somatotrophs. In cells exposed to 1-10 nM hGRF-(1-29) or -(1-44), GH release and 45Ca2+ efflux increased during the first 15 sec and reached peak values within 75 sec. At lower GRF concentrations, 45Ca2+ efflux still increased within 15 sec while GH secretion commenced 15-30 sec later. hGRF-(1-29) increased GH release and 45Ca2+ efflux also after 30 min preperifusion in a calcium-depleted medium with 0.1 mM EGTA added during the last 5 min of preperifusion. However, the magnitude of the stimulation was lower than in the presence of calcium. IBMX increased GH release and 45Ca2+ efflux within 15 sec and peak values were reached within 60 sec. (Bu)2cAMP increased GH release both in the presence and absence of extracellular calcium although the magnitude of stimulation was less in the calcium-depleted medium. Efflux of 45Ca2+ was stimulated by (Bu)2cAMP, independently of extracellular calcium. When exposed to 50 mM K+, both GH release and 45Ca2+ efflux increased within 15 sec and reached high peak values within 60 sec, an effect blocked by removal of extracellular calcium. We conclude that GRF and three other GH secretagogues, (Bu)2cAMP, IBMX, and a high extracellular K+ concentration, rapidly increase 45Ca2+ efflux. GH release and 45Ca2+ efflux appear to be tightly coupled with the calcium response perhaps slightly preceding GH release. This tight coupling strengthens the hypothesis that increased Ca2+ activity is directly involved in exocytosis. GRF and (Bu)2cAMP stimulate GH release and 45Ca2+ efflux also in a calcium-free medium, suggesting that mobilization of intracellular calcium is involved in the action of these secretagogues.
1-Methyl-3-isobutylxanthine↗
Effect of hypophysectomy and growth hormone substitution on red blood cell morphology and filterability in rats.
Growth factors, including insulin, are known to stimulate erythroid cell formation in vitro. This somatotrophic effect of insulin might be involved in blood abnormalities in diabetes mellitus. We therefore investigated whether growth hormone (GH) affects the morphology or filterability of circulating red blood cells (RBCs) in hypophysectomized rats. After hypophysectomy, rats became anemic with a decrease in hematocrit, hemoglobin concentration, RBC count, and reticulocytes. The RBC membrane area was enlarged, giving an increased area-to-volume ratio compared with the control RBCs. The calculated minimum cylindrical diameter (MCD) and resistance to initial folding of the RBC decreased after hypophysectomy, and was confirmed by an enhanced filtration of RBCs through 3 microns Nucleopore membranes. The number of filter clogging particles was also lower in hypophysectomized rats than in control rats. Substitution for 21 days with bovine GH (via subcutaneously implanted osmotic minipumps) normalized RBC morphology and filterability. After withdrawal of GH, all parameters returned toward the presubstitutional levels. Neither cortisone nor thyroxine had any measurable effect on RBC morphology or deformability, indicating that GH deficiency contributes to the abnormal RBC morphology and rheology seen after hypophysectomy.
Animals↗
Growth hormone-releasing factor induces a biphasic modulation of K+ permeability in perfused cultured rat somatotrophs.
Growth hormone-releasing factor (GRF) induced a biphasic response in 86Rb outflow in rat somatotrophs. An early inhibition was observed at all GRF concentrations tested, I pM-10 nM, and this was followed by a later increase. The increase in 86Rb outflow rate came earlier at high GRF concentrations and GRF had a dose-related effect on the magnitude of the increase. The effects of GRF on 86Rb outflow were observed also in the absence of extracellular calcium. Peak growth hormone release preceded peak 86Rb outflow rate. The timing of the effects of GRF on potassium outflow is consistent with the hypothesis that reduced potassium outflow is involved in somatotroph depolarization leading to increased exocytosis, followed by a later increase in potassium outflow leading to repolarization.
Animals↗
Perchlorate stimulates potassium-induced growth hormone release in cultured rat somatotrophs.
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Animals↗
An ultrastructural and functional characterization of rat somatotrophs highly enriched on a continuous Percoll density gradient.
We here describe the use of a continuous Percoll density gradient in preparing a fraction of consistently more than 95% rat somatotrophs. Recovery was about 50% and cell viability 98-99.5%. Two groups of somatotrophs were identified, one (74-82%, type II) heavily and another (18-26%, type I) sparsely granulated. Freshly prepared somatotrophs responded to growth hormone-releasing factor but the responsiveness was greatly enhanced after 3 days' culture. Electron microscopy revealed a well-preserved morphological integrity in both freshly prepared and cultured somatotrophs. culture reduced the average granule profile area and granule volume density of type II cells. This population of pure and well-functioning somatotrophs is suited for studies on the cellular mechanisms involved in growth hormone secretion.
Animals↗
Non-palpable inguinal hernia in the female.
A total of 142 inguinal hernioplasties in 130 female patients with nonpalpable inguinal hernias were performed over a period of 8 years. The mean age in this series was 32 years. (Range 10-76 years). One hundred thirty six cases were followed 3-51 months postoperatively. One hundred seventeen of these (86%) were considered to have good results. Nonpalpable inguinal hernia in the female is clinically recognizable on the basis of intermittency, character and localization of the pain and typical findings at the clinical examination. About three quarters of these patients report in addition to dull inguinal pain, intermittent neuralgic pain and in almost two thirds of the patients a pin-prick hyperalgesia of the skin corresponding to the ilio-inguinal nerve can be demonstrated. It is also typical that all patients experience a distinct tenderness upon palpation over the deep inguinal ring during Valsalva's maneuvre. It is important to keep this condition in mind, especially since the patients respond well to surgical treatment.
Adolescent↗