Alpha-mannosidosis: characterization of CNS pathology and correlation between CNS pathology and cognitive function.

Alpha-mannosidosis (AM) (OMIM 248500) is a rare lysosomal storage disease. The understanding of the central nervous system (CNS) pathology is limited. This study is the first describing the CNS pathology and the correlation between the CNS pathology and intellectual disabilities in human AM. Thirty-four patients, aged 6-35 years, with AM were included. Data from 13 healthy controls were included in the analysis of the magnetic resonance spectroscopy (MRS). Measurements of CNS neurodegeneration biomarkers in cerebrospinal fluid (CSF), CSF-oligosaccharides, and performance of cerebral magnetic resonance imaging (MRI) and MRS were carried out. On MRI, 5 of 10 patients had occipital white matter (WM) signal abnormalities, and 6 of 10 patients had age-inappropriate myelination. MRS demonstrated significantly elevated mannose complex in gray matter and WM. We found elevated concentrations of tau-protein, glial fibrillary acidic protein and neurofilament light protein in 97 patients, 74% and 41% of CSF samples, respectively. A negative correlation between CSF-biomarkers and cognitive function and CSF-oligosaccharides and cognitive function was found. The combination of MRS/MRI changes, elevated concentrations of CSF-biomarkers and CSF-oligosaccharides suggests gliosis and reduced myelination, as part of the CNS pathology in AM. Our data demonstrate early neuropathological changes, which may be taken into consideration when planning initiation of treatment.

Cognitive profile and activities of daily living: 35 patients with alpha-mannosidosis.

BACKGROUND: Alpha-mannosidosis (OMIM 248500) (AM) is a rare lysosomal storage disease caused by a deficiency of the alpha-mannosidase enzyme. The typical signs consist of hearing impairment, intellectual disabilities, coarse facial features and motor function disturbances. We report on the cognitive function and activities of daily living in patients with AM. METHODS: Thirty five AM patients, age 6-35 years, were included in the study. As a cognitive function test, we used the Leiter international performance scale-revised (Leiter-R), which consists of two batteries: the visual function and reasoning battery and the memory and attention battery, the latter including a memory screening. Additional two questionnaires, The Childhood Health Assessment Questionnaire (CHAQ) and EQ-5D-5 L, were filled out. RESULTS: We found IQ in the range of 30-81 in our cohort. The total equivalent age (mental age) was significantly reduced, between 3-9 years old for the visual function and reasoning battery, between 2.3-10.2 years for the memory screening. Data suggested a specific developmental profile for AM with a positive intellectual development until the chronological age 10-12 years, followed by a static or slightly increasing intellectual level. All patients were to varying degrees socially and practically dependent and unable to take care of themselves in daily life. CONCLUSIONS: Intellectual disability is a consistent finding in patients with alpha-mannosidosis but with extensive variation. We assess that this group of patients has, despite their intellectual disabilities, a potential for continuous cognitive development, especially during childhood and early teenage years. This should be included and supported in the individual educational planning.

Enzyme replacement therapy for alpha-mannosidosis: 12 months follow-up of a single centre, randomised, multiple dose study.

BACKGROUND: Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disease (LSD) caused by alpha-mannosidase deficiency. Manifestations include intellectual disabilities, facial characteristics and hearing impairment. A recombinant human alpha-mannosidase (rhLAMAN) has been developed for weekly intravenous enzyme replacement therapy (ERT). We present the preliminary data after 12 months of treatment. METHODS: This is a phase I-II study to evaluate safety and efficacy of rhLAMAN. Ten patients (7-17 y) were treated. We investigated efficacy by testing motor function (6-minutes-Walk-Test (6-MWT), 3-min-Stair-Climb-Test (3-MSCT), The Bruininks-Oseretsky Test of Motor Proficiency (BOT2), cognitive function (Leiter-R), oligosaccharides in serum, urine and CSF and Tau- and GFA-protein in CSF. RESULTS: Oligosaccharides: S-, U- and CSF-oligosaccharides decreased 88.6% (CI -92.0 -85.2, p < 0.001), 54.1% (CI -69.5- -38.7, p < 0,001), and 25.7% (CI -44.3- -7.1, p < 0.05), respectively. Biomarkers: CSF-Tau- and GFA-protein decreased 15%, p < 0.009) and 32.5, p < 0.001 respectively. Motor function: Improvements in 3MSCT (31 steps (CI 6.8-40.5, p < 0.01) and in 6MWT (60.4 m (CI -8.9 -51.1, NS) were achieved. Cognitive function: Improvement in the total Equivalence Age of 4 months (0.34) was achieved in the Leiter R test (CI -0.2-0.8, NS). CONCLUSIONS: These data suggest that rhLAMAN may be an encouraging new treatment for patients with alpha-mannosidosis.The study is designed to continue for a total of 18 months. Longer-term follow-up of patients in this study and the future placebo-controlled phase 3 trial are needed to provide greater support for the findings in this study.

A direct comparison of the pharmacodynamic properties of insulin detemir and neutral protamine lispro insulin in patients with type 1 diabetes.

AIMS: To compare the pharmacodynamic properties of insulin detemir (detemir) and neutral protamine lispro (NPL) insulin using a euglycaemic glucose clamp. METHODS: In a double-blind, crossover study, 30 patients with C-peptide negative type 1 diabetes were randomly assigned to a single dose (0.4 U/kg) of detemir and NPL. Plasma glucose (PG) was normalized with a variable insulin infusion and then decreased stepwise, followed by a euglycaemic clamp at 5.5 mmol/l over 32 h. Duration of action was defined as time from dosing until PG exceeded 8.3 mmol/l for at least 30 min. RESULTS: Duration of action was similar for detemir [23.0 (range 2.25-32) h] and NPL [22.0 (9.5-32) h], p = 0.55. Using glucose infusion rate (GIR) parameters, detemir showed a flatter pharmacodynamic profile versus NPL: area under the curve, AUC(GIR) ((0-32)) = 1326 vs. 1841 mg/kg, p < 0.01 (detemir vs. NPL, respectively); AUC(GIR) ((0-12)) = 784 vs. 1392 mg/kg, p < 0.05; AUC(GIR) ((12-32)) = 455 vs. 274 mg/kg, p = 0.051; GIR(late) (12-32)/GIR(early) (0-12) ratio = 0.33 vs. 0.04, p < 0.001. Detemir also showed a lower and later peak of action than NPL [GIR(max) 2.0 vs. 3.2 mg/kg/min, p < 0.01; T(max) 9.1 (95% confidence interval: 3.0-14.7) vs. 7.0 h (1.8-15.2)]. CONCLUSIONS: Detemir and NPL had similar duration of action of approximately 24 h in patients with type 1 diabetes. Compared with NPL, detemir had a flatter profile with a more even distribution of metabolic effect over 24 h.

Lyme neuroborreliosis in 2 horses.

Lyme neuroborreliosis--characterized as chronic, necrosuppurative to nonsuppurative, perivascular to diffuse meningoradiculoneuritis--was diagnosed in 2 horses with progressive neurologic disease. In 1 horse, Borrelia burgdorferi sensu stricto was identified by polymerase chain reaction amplification of B burgdorferi sensu stricto-specific gene targets (ospA, ospC, flaB, dbpA, arp). Highest spirochetal burdens were in tissues with inflammation, including spinal cord, muscle, and joint capsule. Sequence analysis of ospA, ospC, and flaB revealed 99.9% sequence identity to the respective genes in B burgdorferi strain 297, an isolate from a human case of neuroborreliosis. In both horses, spirochetes were visualized in affected tissues with Steiner silver impregnation and by immunohistochemistry, predominantly within the dense collagenous tissue of the dura mater and leptomeninges.

Kinetics, mechanism, and thermochemistry of the gas-phase reaction of atomic chlorine with pyridine.

A laser flash photolysis-resonance fluorescence technique has been employed to study the kinetics of the reaction of atomic chlorine with pyridine (C(5)H(5)N) as a function of temperature (215-435 K) and pressure (25-250 Torr) in nitrogen bath gas. At T> or = 299 K, measured rate coefficients are pressure independent and a significant H/D kinetic isotope effect is observed, suggesting that hydrogen abstraction is the dominant reaction pathway. The following Arrhenius expression adequately describes all kinetic data at 299-435 K for C(5)H(5)N: k(1a) = (2.08 +/- 0.47) x 10(-11) exp[-(1410 +/- 80)/T] cm(3) molecule(-1) s(-1) (uncertainties are 2sigma, precision only). At 216 K < or =T< or = 270 K, measured rate coefficients are pressure dependent and are much faster than computed from the above Arrhenius expression for the H-abstraction pathway, suggesting that the dominant reaction pathway at low temperature is formation of a stable adduct. Over the ranges of temperature, pressure, and pyridine concentration investigated, the adduct undergoes dissociation on the time scale of our experiments (10(-5)-10(-2) s) and establishes an equilibrium with Cl and pyridine. Equilibrium constants for adduct formation and dissociation are determined from the forward and reverse rate coefficients. Second- and third-law analyses of the equilibrium data lead to the following thermochemical parameters for the addition reaction: Delta(r)H = -47.2 +/- 2.8 kJ mol(-1), Delta(r)H = -46.7 +/- 3.2 kJ mol(-1), and Delta(r)S = -98.7 +/- 6.5 J mol(-1) K(-1). The enthalpy changes derived from our data are in good agreement with ab initio calculations reported in the literature (which suggest that the adduct structure is planar and involves formation of an N-Cl sigma-bond). In conjunction with the well-known heats of formation of atomic chlorine and pyridine, the above Delta(r)H values lead to the following heats of formation for C(5)H(5)N-Cl at 298 K and 0 K: Delta(f)H = 216.0 +/- 4.1 kJ mol(-1), Delta(f)H = 233.4 +/- 4.6 kJ mol(-1). Addition of Cl to pyridine could be an important atmospheric loss process for pyridine if the C(5)H(5)N-Cl product is chemically degraded by processes that do not regenerate pyridine with high yield.

Analgesic efficacy and safety of lornoxicam quick-release formulation compared with diclofenac potassium: randomised, double-blind trial in acute low back pain.

BACKGROUND: NSAIDs are widely used for patients presenting with low back pain. A quick-release formulation of lornoxicam, a potent NSAID from the chemical class of oxicams, offers a faster onset of pain relief compared with the standard tablet formulation. METHODS: Time to onset of pain relief with lornoxicam was compared with the quick-release formulation of diclofenac potassium in acute low back pain in a randomised, double-blind, multicentre study. 220 patients received either lornoxicam 24 mg or diclofenac potassium 150 mg on day 1 followed by lornoxicam 8 mg twice daily or diclofenac potassium 50 mg twice daily for 5 days. Efficacy outcomes included time to onset of pain relief, as measured by the stopwatch method (primary outcome), pain intensity, pain relief, rescue medication, ability to perform daily activities and global evaluation of the study medication. RESULTS: The time to onset of pain relief ratios between diclofenac potassium/lornoxicam was 1.03 (95% CI 0.91, 1.26) and 1.05 (95% CI 0.93, 1.29) in the intention-to-treat (ITT) and per-protocol (PP) analyses, respectively, demonstrating the non-inferiority of lornoxicam (defined by lower limits of the 95% CIs >0.80). Time to onset of pain relief was shorter with lornoxicam (30 minutes) compared with diclofenac potassium (36 minutes). The difference was not statistically significant (ITT analysis). A higher magnitude of analgesic effect associated with better global evaluation of the study medication for lornoxicam was also demonstrated. The drugs were equally well tolerated. CONCLUSION: Lornoxicam administered as a quick-release formulation was shown to be non-inferior to the equivalent formulation of diclofenac potassium in terms of onset of pain relief and more effective on most of the major standard efficacy outcomes.

Validity and use of a non-parallel insulin assay for pharmacokinetic studies of the rapid-acting insulin analogue, insulin aspart.

A radioimmunoassay (RIA) for insulin was validated for reliable measurement of the human insulin analogue, insulin aspart, by correction of non-linear measurements. Specificity was equivalent for several species of insulin, except insulin aspart. A non-linear hyperbolic model fitted insulin aspart with a correction formula for non-linearity of: z = 1,503y/ (1,398 - y), where y denotes measured concentration and z denotes true concentration. Matrix-effects were insignificant for human, porcine, and canine heparin-plasma and for human and porcine serum. The coefficient of variation was below 15% for 80-800 pmol/L human and porcine insulin and for 80-600 pmol/L insulin aspart. The limit of detection for insulin aspart was 11.5 pmol/L with a lower limit of quantification of 17.5 pmol/ L. Dilution of serum with Pharmacia dilution media introduced no significant error. In conclusion, this paper demonstrates that a non-parallel radioimmunoassay can be used to estimate accurate concentrations of insulin aspart.

Genetic immunotherapy by intrapleural, intraperitoneal and subcutaneous injection of IL-2 gene-modified Lewis lung carcinoma cells.

The induction and augmentation of tumor non-specific immunity and of tumor-specific immunity by intrapleural, intraperitoneal and subcutaneous injection of interleukin-2 (IL-2) gene-modified Lewis lung carcinoma (LLC) cells (LLC-IL2) was tested in C57BL/6 mice. Intrapleural injection of LLC cells induced lung tumors with a malignant effusion, intraperitoneal injection induced peritoneal tumors with ascites and subcutaneous injection induced subcutaneous tumors. Intrapleural injection of irradiated LLC-IL2 cured pre-existing lung LLC tumors and extended the survival of the mice but did not affect survival of mice with pre-existing peritoneal tumors nor did it affect the growth of s.c. tumors. Intraperitoneal injection of irradiated LLC-IL2 cured pre-existing LLC peritoneal tumors and extended the survival of the mice but did not affect survival of mice bearing lung tumors nor did it affect the growth of s.c. tumors. Subcutaneous injection of irradiated LLC-IL2 did not affect the growth of preexisting s.c. tumors and also did not improve survival of mice bearing the lung or peritoneal tumors. Injection with irradiated LLC-IL2 by all routes, i.e., intrapleural, intraperitoneal and s.c., protected against subsequent re-challenge with LLC. Eight days after the initial immunization (early stage of immunization), non-adherent mononuclear cells in the peritoneal cavity of the mice treated with intraperitoneal injection of irradiated LLC-IL2 displayed enhanced cytotoxicity against LLC, B16-F10 and P815 cells, while the cytotoxic activity of spleen cells in the same mice did not change. The efficiency of induction of tumor-specific immunity was the strongest after intraperitoneal immunization and weakest after s.c. immunization. In vitro analysis using the spleen cells of mice immunized with irradiated LLC-IL2 suggested that CD8+ T cells play a key role in tumor-specific immunity.

Pulsed dose rate (PDR) brachytherapy of anal carcinoma.

BACKGROUND AND PURPOSE: With radiotherapy of anal carcinomas, sphincter preservation can be obtained at survival rates similar to those obtained with radical surgery. By combining external beam irradiation with interstitial irradiation, superiority over standard external irradiation has been obtained. With the introduction of pulsed dose rate equipment, where a single high activity source moves through catheters, a more individualized dose distribution and a further elimination of radiation exposure to the staff can be achieved. MATERIALS AND METHODS: Between June 1993 and November 1994, 17 patients with anal carcinoma (T1:4, T2:4, T3:6, T4:3) have been treated at the Finsen Center. The treatment consisted of three-field external irradiation 46 Gy/23 fractions with five fractions a week to the anal canal and regional pelvic lymph nodes. Seven to 33 days after completion of external irradiation, the tumorspace was given 25.2 Gy PDR brachytherapy with 42 pulses of 0.6 Gy, one pulse every hour. RESULTS: One isolated local recurrence has been noted 13 weeks after implantation. One additional local recurrence was seen in a patient with concomitant hepatic and inguinal recurrence. In three patients inguinal recurrence had occurred, two of these patients were irradiated without any further evidence of disease, and one patient with a primary advanced tumour, had local failure. So far necrosis has been observed in 13 patients within 1-49 weeks (median 16 weeks) after implantation. Eight of these patients required colostomy. No relation was observed between the number of implanted needles and the occurrence of necrosis. CONCLUSIONS: The results indicate that the treatment is highly effective, but with substantial toxicity.

Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

Analysis of perforin expression in human peripheral blood lymphocytes, CD56+ natural killer cell subsets and its induction by interleukin-2.

In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin positive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25-30% of this population represents the CD56bright+ subset, while 70-75% represents the CD56dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97-99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin positive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2.

Perforin expression by thyroid-infiltrating T cells in autoimmune thyroid disease.

Infiltration of the thyroid gland by lymphocytes is a hall-mark of autoimmune thyroid disease; it is particularly evident in Hashimoto's thyroiditis but is also seen in most patients with Graves' disease. Infiltrating cells are comprised primarily of T lymphocytes, of which only a minority appears to be activated. Their precise pathogenic role is largely unknown. Since perforin has been a marker for functionally activated cytotoxic T cells in situ we elected to assess the presence of perforin-containing cells in thyroid-infiltrating lymphocytes and establish their phenotype. Cells were isolated from seven subtotal thyroidectomy specimens, five from patients with Graves' disease and two with Hashimoto's thyroiditis. The novel findings were as follows: CD4+ perforin-containing T cells occurred only in Hashimoto's glands, suggesting a class II-restricted component of cytotoxicity; in Graves' disease, and to a lesser extent in Hashimoto's, perforin-expressing cells were primarily T cell receptor alpha beta- CD4-CD8- (double negative); double negative perforin-containing cells in peripheral blood of normal individuals were largely gamma delta + T cells. In Hashimoto's samples, the predominant population of T cells expressing perforin was CD8+. By comparison, in studies of the synovial fluid of knee joints from patients with rheumatoid arthritis only a minor population of the perforin-containing cells was double-negative. The data suggest significant differences in cytotoxic autoimmune mechanisms between the two autoimmune thyroid diseases. Functional characterization of double-negative T cells is necessary to define their role in autoimmunity.

Gene therapy for Lewis lung carcinoma with tumor necrosis factor and interleukin 2 cDNAs co-transfected subline.

Gene therapy with cytokine cDNA will provide a new tool for cancer treatment. We have already reported that immunization with interleukin-2 (IL2) cDNA transfected Lewis lung carcinoma (LLC) cells induced anti-tumor immunity, which, however, was not strong enough to eradicate an established tumor. In an attempt to develop more effective gene therapy methods, we have used tumor cells co-transfected with IL-2 and tumor necrosis factor (TNF) cDNAs. These cDNAs were introduced into pBMG-Neo and pcDV-X819 vectors, respectively, and then co-transfected into LLC cells. The co-transfectants were selected by incubating them in a medium containing G418 followed by the limiting dilution method twice to obtain IL2 and TNF cDNA co-transfected LLC (LLC-TNF-IL2) cells. When 5 x 10(5)/ml LLC-TNF-IL2 cells were incubated for 48 h, they secreted 7.56 U/ml TNF and 527.0 U/ml IL2 into the culture supernatant. When C57BL/6 mice were transplanted with 1 x 10(6) LLC-TNF-IL2 cells, all the tumors were rejected. The growth of transplanted LLC, but not B16F10 melanoma cells, was retarded in mice inoculated with LLC-TNF-IL2 on their contralateral sides, which suggests specific immunity was induced. The immunization effect by the co-transfectant was superior to that of the IL2- and TNF-transfectants alone.

Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice.

Perforin-deficient mice have been generated by homologous recombination to determine whether the effects of CD8+ cytolytic T cells and natural killer cells are mediated by pore formation involving perforin. These mice are viable and fertile and have normal numbers of CD8+ T cells and natural killer cells which do not lyse virus-infected or allogeneic fibroblasts or natural killer target cells in vitro. The mice fail to clear lymphocytic choriomeningitis virus and they eliminate fibrosarcoma tumour cells with reduced efficiency. Perforin is therefore a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis.

In vitro and in vivo growth of B16F10 melanoma cells transfected with interleukin-4 cDNA and gene therapy with the transfectant.

In an attempt to develop the most effective cytokine gene therapy, we transfected mouse interleukin(IL)-2, mouse IL-4, and human IL-6 cDNAs into mouse melanoma cells, B16F10. Transfection with IL-4 cDNA decreased the tumorigenicity of B16F10 most strongly. We investigated whether gene therapy with IL-4-transfected B16F10 cells was possible. Flow-cytometric analysis showed that major histocompatibility complex class I and II expression in B16F10 and IL-4-cDNA-transfected B16F10 (B16F10-IL4) cells did not differ. Doubling times of B16F10 and B16F10-IL4 were 20.1 and 21.1 h respectively. The growth of B16F10 cells was retarded if C57BL/6 mice were inoculated with B16F10-IL4 at the contralateral sides. When 5 x 10(5) B16F10 cells were transplanted subcutaneously into the flanks of C57BL/6 mice, they all developed a tumor mass, whereas no tumor masses formed in those transplanted with B16F10-IL4 cells within 60 days. No nude, severe combined immunodeficient or beige mice were able to reject parental B16F10 or B16F10-IL4 cells, although, B16F10-IL4 tumor growth in all these immunodeficient mice was slower than that of B16F10. Therefore, we concluded that T and natural killer cells are necessary for rejection of B16F10-IL4 tumor cells.

Functional effects of CD30 on a large granular lymphoma cell line, YT. Inhibition of cytotoxicity, regulation of CD28 and IL-2R, and induction of homotypic aggregation.

Studies are described revealing novel regulatory functions for the lymphocyte activation Ag CD30. A new mAb, C10, reactive with YT cells binds to CD30 and induces inhibition of the cytotoxicity of YT for Raji cells. C10 inhibition of cytotoxicity requires several hours preincubation of YT with C10; the antibody has no effect if added directly to YT cytotoxicity assays. CD30 stimulation by C10 down-regulates CD28 expression on YT by > 80% within 48 h. Because CD28 is required for YT cytotoxicity toward Raji cells and other B7/BB1 bearing targets, it is suggested that inhibition of cytotoxicity of YT is mediated by control of CD28 expression and/or signaling via CD30. Accordingly, conjugation of YT with Raji is only slightly affected by CD30-mediated down-regulation of CD28, and perforin mRNA steady state levels are not changed at all. C10 treatment of YT cells additionally down-regulates the expression CD45 and up-regulates IL-2R p55. Moreover, CD30 stimulation by C10 causes homotypic aggregation of YT. Homotypic aggregation is slow, requiring gene transcription, translation, metabolic energy at elevated temperature (37 degrees C), magnesium ions, and an intact cytoskeleton. These studies offer insights into the function of CD30 as a complex regulator of T cells.

Interleukin-6 cDNA transfected Lewis lung carcinoma cells show unaltered net tumour growth rate but cause weight loss and shortened survival in syngeneic mice.

HuIL-6 cDNA, cloned into a neomycin resistant conferring expression vector, BMGNeo, was transfected into Lewis Lung Carcinoma (LLC) cells. LLC cells (5 x 10(6) ml-1) transfected with IL-6 cDNA (LLC-IL6) secreted IL-6 into the culture supernatant at a concentration of 9.9 ng ml-1 within 48 h. When 1,000,000 of untransfected LLC, BMGNeo vector transfected LLC (LLC-Neo) or LLC-IL6 cells were transplanted into C57BL/6 mice subcutaneously, the mean +/- s.d. of survival times of these mice were 33.3 +/- 9.7, 34.3 +/- 7.1 and 17.0 +/- 3.1 days, respectively. The survival time of LLC-IL6 cells transplanted mice was significantly shorter than that of LLC (P < 0.01) or LLC-Neo (P < 0.01) cells transplanted mice without a measurable difference of tumour size. Plasma concentration of IL-6 steadily increased in LLC-IL6 transplanted mice. Body weight and serum albumin were significantly lower in LLC-IL6 transplanted mice than in LLC transplanted mice. Mouse IL-1 alpha and mouse TNF-alpha were not detected in the plasma of LLC-IL6 transplanted mice. These data suggested that secretion of IL-6 from LLC cells was unable to alter net tumour growth rate but rather caused a state similar to cachexia without detectable increase of IL-1 alpha and TNF-alpha in the plasma. This state may be responsible for the shortened survival of LLC-IL6 tumour-bearing mice.

Combination effect of vaccination with IL2 and IL4 cDNA transfected cells on the induction of a therapeutic immune response against Lewis lung carcinoma cells.

In order to develop a more effective method of immunotherapy we have transfected mouse interleukin-2 (IL2) or mouse interleukin-4 (IL4) cDNA into a spontaneous non-immunogenic murine lung cancer. Lewis lung carcinoma (LLC). IL2 cDNA transfection more strongly decreases tumorigenicity of LLC than IL4 cDNA transfection. Recombinant-human-IL2 treatment of mice that were transplanted with untransfected LLC could not prolong their survival. In contrast, vaccination with IL2-cDNA-transfected LLC (LLC-IL2) and LLC-IL2 mixed with IL4-cDNA-transfected LLC (LLC-IL4) could significantly suppress tumor growth of LLC in a tumor-specific manner. The vaccination with LLC-IL2 mixed with the same number of LLC-IL4 cells was more suppressive to the growth of LLC than that with LLC-IL2 cells alone, while LLC-IL4 vaccination alone was ineffective. Nude, severe-combined-immune-deficient (SCID) and beige mice were unable to reject LLC-IL2 cells. However, immunodeficient mice responded to LLC-IL2, but not to LLC, since their survival times after transplantation with LLC-IL2 cells were significantly longer than the survival time of normal or immunodeficient mice transplanted with untransfected LLC cells. We conclude that vaccination with IL2-producing tumors and, with more pronounced effect, in combination with IL4-producing tumors, is able to induce an immune response to this normally non-immunogenic tumor. Tumor rejection appears to be achieved by the combined activity of CTL and NK cells. This strategy has potential for new immunotherapeutic interventions in cancer patients.

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.