Gene therapy with autologous, interleukin 2-secreting tumor cells in patients with malignant melanoma.

We vaccinated metastatic melanoma patients with irradiated, autologous melanoma cells genetically engineered to secrete interleukin 2 (IL-2) to investigate whether an anti-tumor immune response would be induced. Melanoma cell cultures were established from surgical specimens and were engineered to secrete IL-2 by infection with recombinant retrovirus. Twelve patients were vaccinated subcutaneously one, two, or three times with approximately 10(7) irradiated, autologous, IL-2-secreting tumor cells. Treatment was well tolerated, with local reactions at 11 of 24 injection sites and minor systemic symptoms of fever and headache after 6 injections. One patient developed anti-tumor DTH after the first vaccination and showed an increased response after the second vaccination. Anti-autologous tumor CTLs could be detected prevaccination in the peripheral blood of seven patients and their activity increased after vaccination in four patients. No UICC-defined clinical responses were seen, but three patients had stable disease for 7-15 months, one of whom has not yet progressed (15+ months). Thus, patient vaccination with autologous, genetically engineered tumor cells is feasible and safe. Anti-tumor DTH and CTLs can be induced in some patients with such a vaccine.

A cellulose acetate immunofixation technique.

A method of immunofixation using CAM as the only substrate is described. It consists of separating the proteins by conventional electrophoresis followed by immersion for a few minutes only in appropriate specific antiserum suitably diluted with barbitone buffer containing polyethylene glycol (PEG). This is followed by washing and staining by Ponceau S or Nigrosin. The advantages of the method used are speed, sensitivity and economy, no expensive equipment or reagents being required. The method has proved helpful in the identification and classification of minor paraprotein bands and in the analysis of multiclonal paraproteinaemias. The technique can be applied as well to other proteins of biological fluids, e.g. phenotyping.

Contaminant bands on SDS-polyacrylamide gel electrophoresis are recognised by antibodies in normal human serum and saliva.

Contamination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis samples has commonly produced artefactual bands when sensitive detection systems, such as silver staining or immunoblotting procedures, have been used. Evidence suggests that these bands originate from human skin contamination of reagents and equipment. The presence of antibodies to contaminating bands, in serum and saliva samples from normal donors, detected in immunoblotting experiments, is described; the isotype distribution of these naturally occurring antibodies has also been investigated.

The use of alkaline-phosphatase conjugated second antibody for the enhancement of immunoprecipitation techniques on cellulose acetate membranes.

A technique is described using alkaline phosphatase (ALP) conjugated second antibodies on cellulose acetate membrane (CAM) for enhancement of immunoprecipitation techniques. The method is extremely sensitive, detecting as little as 0.1 mg/l of protein and has particular application in the investigation of monoclonal immunoglobulins in human serum or urine and in hybridoma supernatants or ascitic fluids.

Combined detection of phenotype and Y chromosome by immunoenzyme labelling and in situ hybridisation on peripheral lymphocytes.

A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convenient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.

Asbestosis. Bronchoalveolar lavage fluid proteins and their relationship to pulmonary epithelial permeability.

We measured levels of albumin and immunoglobulins in serum and bronchoalveolar lavage (BAL) fluid in 28 men with asbestosis and 11 control subjects. The half-time clearance of inhaled diethylene triamine pentacetate labelled with technetium-99m (99mTc-DTPA) from the lungs (t1/2LB) was measured in 26 patients with asbestosis and in 31 normal nonsmoking controls. In those individuals in whom immunoglobulins were detected in BAL fluid, the mean IgG:albumin ratio in BAL fluid was 0.30 (range, 0.11 to 0.97), significantly less than the ratio of 0.43 (0.28 to 0.66) in control subjects (p less than 0.05). There was no significant difference in IgA:albumin ratios between patients and control subjects. The mean BAL:serum albumin ratio in patients with asbestosis was 2.3 X 10(-3) (range, 0.2 to 9.5 X 10(-3), significantly greater than the ratio of 1.2 X 10(-3) (0.5 to 2.0 X 10(-3] in control subjects (p less than 0.02). The t1/2LB was significantly shorter in both smokers and nonsmokers with asbestosis, compared with 31 normal nonsmoking controls, but there were no relationships between t1/2LB and BAL:serum albumin ratio or any other BAL protein levels in either smokers or nonsmokers with asbestosis.

Monitoring cytokine production in peripheral blood during acute graft-versus-host disease following allogeneic bone marrow transplantation.

Plasma concentrations and peripheral blood cells containing cytoplasmic cytokines were monitored during the post-transplant period in 10 patients who had received allogeneic bone marrow transplants (BMT) for the correction of inherited genetic disorders. The presence of CD14-positive cells containing cytoplasmic interleukin-1 alpha and beta in the peripheral blood was indicative of acute graft-versus-host disease (GVHD). Plasma concentrations of IL-1 alpha, IL-1 beta and TNF-alpha were significantly raised in the GVHD group when compared with the uneventful days. There was, however, poor temporal correlation between the plasma concentrations and clinical manifestations of acute GVHD. Cells containing cytoplasmic IL-6 were present in the peripheral blood when patients had clinically suspected and/or microbiologically confirmed infection. The results from this study demonstrate that analysis of peripheral blood cells for cytoplasmic IL-1 alpha and IL-1 beta are better markers of acute GVHD than is monitoring plasma concentrations of these cytokines.

C3 and C4 complement components and acute phase proteins in late pregnancy and parturition.

To determine whether changes in complement or acute phase proteins can predict onset of labour, complement components C3 and C4, alpha-1-antitrypsin, and alpha-1-acid glycoprotein were measured serially in the third trimester of pregnancy and throughout labour in 11 women. C-reactive protein (CRP) concentration, C3 conversion products, and factor B activation was studied in five pregnancies one week before delivery, during labour, and six weeks after delivery. C3, C4, and CRP concentrations were unchanged and there was no evidence of complement conversion, either immediately before or during labour, by the classical or alternative pathways. A wide variation among subjects but a small variation within subjects for all proteins assayed was noted. It is important to use serial rather than cross sectional biochemical data when determining normal physiological patterns in pregnancy. The proteins measured have no predictive value in the timing of the onset of normal labour.

Identification of the paraproteins and clinical significance of more than one paraprotein and clinical significance of more than one paraprotein in serum of 56 patients.

Sera from 56 patients with more than one paraprotein were investigated for immunoglobin class and light chain type of each paraprotein. The patients were divided into two groups according to the diagnosis, that is myeloma and macroglobulinaemia or others. The frequency of combinations of paraproteins was considered in the whole series and in the two groups. The laboratory and clinical findings were analysed to investigate the diagnostic and prognostic significance of more than one paraprotein in a serum. It is concluded that a lower percentage of patients with more than one paraprotein can definitely be shown to have myeloma than might be expected from studies on monoclonal paraproteinaemia, that patients having IgA paraproteins in the serum had the poorest prognosis, and that paraproteins with lambda light chains were more likely to be associated with myeloma or macroglobulinaemia. A discriminant analysis of ESR and total paraprotein levels in the two groups of patients showed that combinations of the two parameters were not more effective at distinguishing the groups than the ESR alone.

Paraproteinaemia in neurological disease: incidence, associations, and classification of monoclonal immunoglobulins.

Fifty-six patients presenting with neurological complaints were found to have paraproteinaemia unassociated with immunocytic malignancy; 16 patients presented with peripheral neuropathy. There was an association between IgM paraproteinaemia and an idiopathic neuropathy with markedly slowed nerve conduction velocities.

Antibody synthesis within the central nervous system: comparisons of CSF IgG indices and electrophoresis.

VARIOUS LABORATORIES HAVE REPORTED DIFFERING SUCCESS RATES IN THEIR ABILITY TO DETECT INTRATHECAL SYNTHESIS OF ANTIBODY WHEN COMPARING THE INDEX OF [FORMULA: see text] with electrophoretic analysis. We selected 44 patients in the borderline area of minimal and/or equivocal abnormality by IgG index. Electrophoretic analysis (on polyacrylamide gels for oligoclonal gamma globulin pattern) of parallel specimens was performed at the same time. The number of samples giving a normal index but showing oligoclonal bands varied between 34% and 43% depending on the cut-off point. The views about normal barrier functions underlying such indices are discussed with particular reference to the pathophysiology of the blood-CSF barrier.

Clinical applications of immunofixation: detection and quantitation of complement activation.

The methods currently in use for the detection and quantitation of complement activation products are slow and time consuming. We describe a method utilising immunofixation after agarose or cellulose acetate membrane electrophoresis which allows large batches of samples to be screened rapidly for the presence of activation products of C3 and factor B. Further, after immunofixation on agarose the conversion product may be quantitated by densitometry. This method gives similar results to those obtained by crossed immunoelectrophoresis. Using both this technique and crossed immunoelectrophoresis we have been able to confirm that C4 activation occurs during electrophoresis in the absence of EDTA and that in the presence of EDTA it is not demonstrable even in patients with active immune complex disease.

Value of serum C-reactive protein measurement in the management of bone marrow transplant recipients. Part I: Early transplant period.

Serum C-reactive protein concentrations were measured serially during the early transplant period in 68 bone marrow recipients transplanted for leukaemia (34), chronic granulocytic leukaemia (2), severe aplastic anaemia (6), and various inborn errors of metabolism (26). There were 116 clearly documented episodes of infection or acute graft versus host disease or both. Serum C-reactive protein concentrations in patients with viral (11) or fungal infection (6) were normal or only slightly raised. In 32 patients with isolated acute graft versus host disease, only three (10%) showed serum C-reactive protein concentrations above 40 mg/l. Values greater than 40 mg/l were strongly suggestive of bacterial infections and values above 100 mg/l were seen only in patients (43) with bacterial infections with or without acute graft versus host disease. These findings suggest that serum C-reactive protein concentrations are valuable both for diagnosis and monitoring of such infections.

Value of serum C-reactive protein measurement in the management of bone marrow transplant recipients. Part II: Late post-transplant period.

Seventeen bone marrow recipients transplanted for acute leukaemia (8), chronic leukaemia (1), severe aplastic anaemia (3), and various inborn errors of metabolism (5) had 22 episodes of documented infection in the late (greater than 3 months) post-transplant period. Serum C-reactive protein concentrations were considerably increased in patients with bacterial infections, but not in those with viral or fungal infections. Serum C-reactive protein values were normal in 20 patients transplanted for acute leukaemia (12), chronic leukaemia (1), severe aplastic anaemia (2), and various inborn errors of metabolism (5) who had active chronic graft versus host disease but no evidence of infection. These findings indicate that serum C-reactive protein concentrations are useful in the diagnosis and monitoring of bacterial infections even in the presence of chronic graft versus host disease.

Increased serum IgE concentrations during infection and graft versus host disease after bone marrow transplantation.

Serum IgE concentrations estimated in 25 bone marrow transplant recipients during episodes of infection or graft versus host disease, or both, were raised not only in some patients with acute graft versus host disease but also in many patients with infection. Raised values were not seen in chronic graft versus host disease. The routine estimation of serum IgE in bone marrow transplant recipients had minimal value because of the lack of specificity of the IgE response.

Laboratory investigation of monoclonal gammopathy during 10 years of screening in a general hospital.

Protein electrophoresis was carried out on 102,000 samples from the patients of a district general hospital over 10 years, and a monoclonal protein was detected in 730 cases; of these, 114 could be classified as B cell malignancies and 261 as monoclonal gammopathy of undefined significance (MGUS). The various clinical and laboratory features of monoclonal gammopathy were examined with respect to distinguishing the malignant conditions from MGUS at first presentation.

Cytokine gene expression in skin and lymphoid organs in graft versus host disease.

AIM: To determine if human graft versus host disease (GvHD) is associated with any detectable change in cytokine gene expression in the skin and lymphoid organs. METHODS: Reverse transcriptase and the polymerase chain reaction were used to amplify mRNA for interleukins-1 (IL-1), -2 (IL-2), -4 (IL-4) and -6 (IL-6), IL-2 receptor (IL-2R), tumour necrosis factors alpha (TNF-alpha) and beta (TNF-beta), gamma interferon (IFN gamma) and granulocyte macrophage colony stimulating factor (GM-CSF) in frozen punch biopsy specimens of skin and necropsy samples of skin, lymph node, and spleen. RESULTS: No cytokine mRNA was detected in the punch biopsy specimens except weak signals for IL-6 and IL-1 and GM-CSF in two normal donors and IL2-R in one patient with GvHD. In samples of skin taken at necropsy, however, significant quantities of mRNA for TNF-alpha, TNF-beta, and IL-4 were detected in patients who had or had had GvHD in contrast to those without the disease whose skin lacked mRNA for these products but contained detectable quantities of IL-1, IL2-R, IL-6 and GM-CSF. There seemed to be a reciprocal relation between TNF-alpha and IL-4. In necropsy samples of lymph node and spleen a pattern of cytokine production similar to that in the skin was observed with a preponderance of TNF-alpha, TNF-beta and IL-4 in patients with GvHD and GM-CSF and IL-6 in those without the disease. CONCLUSIONS: The local synthesis of these molecules would explain many of the morphological and immunohistological features of GvHD. The failure to detect TNF-alpha, TNF-beta, and IL-4 in skin biopsy specimens exhibiting GvHD is probably due to their small size but further investigations are required.

The binding of [3H]chlorambucil to nuclear proteins of the Yoshida ascites sarcoma.

The binding of [3H]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of 3H-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.

Some effects of chlorambucil on nuclear protein phosphorylation in the Yoshida ascites sarcoma.

The ability of nuclei, isolated from Yoshida ascites sarcoma cells, to phosphorylate nuclear proteins in the presence of [gamma-32P]ATP has been investigated. Comparisons were made between a strain sensitive to the effects of the alkylating agent, chlorambucil, with a corresponding resistant strain both before and after drug-treatment of tumour-bearing animals. There was no gross quantitative differences between the drug-sensitive and-resistant untreated cells but treatment resulted in increased levels in the sensitive strain. Qualitative differences were seen before treatment in the phosphorylation pattern of the tris-saline-soluble nuclear sap fraction. The high molecular weight species in the fraction from sensitive cells showed phosphorylations which were absent, or present at very low levels, in the corresponding fraction from drug-resistant cells. Changes were observed in the tris-saline-soluble and non-histone protein fractions from drug-sensitive cells following treatment of tumour-bearing animals. Only minor alterations in patterns of phosphorylation were seen in fractions from drug-resistant cells.

Improved resolution of cellulose acetate membrane electrophoresis.

A simple modification to cellulose acetate membrane electrophoresis is described. The results are comparable to those achieved with agarose gels at high voltage.