Immunologic identification of the aromatase enzyme system in human endometrium

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.     

Immunologic responses in patients with pulmonary carcinoma receiving immunotherapy

Summary This is a preliminary report on immune responses observed in vivo and in vitro in resectable patients treated with oral BCG, and in nonresectable patients treated with autologous tumour cells complexed with PPD and with BCG applied by scarification. In some cases, to shorten the period of sensitization, transfer factor was given additionally. Several of the in vitro methods used for assessing the immune competence of the patients are shown to be sufficiently informative to warrant their use before immunotherapy in order to assess the immunologic resources of the patient. During immunotherapy, they allow monitoring the effect of the treatment.     

Hereditary hepatic porphyria with delta aminolevulinate dehydrase deficiency: Immunologic characterization of the non-catalytic enzyme

Summary Immunoreactive -aminolevulinate dehydrase (ALA-D) was measured in lysates from two porphyric patients with ALA-D deficiency (enzyme activities were below 2% of the normal level). By using two different immunologic methods, we found a cross-reactive immunologic material (CRIM+) which corresponded to 20% and 33% of the control level. Therefore the molecular basis that accounts for the deficiency of ALA-D in these patients is a structurally modified enzyme. The methods used to determine the molecular weight (by Western blotting) and the isoelectric point (by chromatofocusing) of the mutants did not show any difference by comparison with the normal enzyme.     

Reduction of elevated plasma globotriaosylsphingosine in patients with classic Fabry disease following enzyme replacement therapy

Fabry disease is treated by two-weekly infusions with α-galactosidase A, which is deficient in this X-linked globotriaosylceramide (Gb3) storage disorder. Elevated plasma globotriaosylsphingosine (lysoGb3) is a hallmark of classical Fabry disease. We investigated effects of enzyme replacement therapy (ERT) on plasma levels of lysoGb3 and Gb3 in patients with classical Fabry disease treated with agalsidase alfa at 0.2 mg/kg, agalsidase beta at 0.2 mg/kg or at 1.0 mg/kg bodyweight. Each treatment regimen led to prominent reductions of plasma lysoGb3 in Fabry males within 3 months (P = 0.0313), followed by relative stability later on. Many males developed antibodies against α-galactosidase A, particularly those treated with agalsidase beta. Patients with antibodies tended towards smaller correction in plasma lysoGb3 concentration, whereas treatment with high dose agalsidase beta allowed a reduction comparable to patients without antibodies. Pre-treatment plasma lysoGb3 concentrations of Fabry females were relatively low. In all females and with each treatment regimen, ERT gave reduction or stabilisation of plasma lysoGb3. Our investigation revealed that ERT of Fabry patients reduces plasma lysoGb3, regardless of the recombinant enzyme used. This finding shows that ERT can correct a characteristic biochemical abnormality in Fabry patients.     

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n=14) and healthy age-matched controls (n=14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.     

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n = 14) and healthy age-matched controls (n = 14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.     

Immunologic studies in asymptomatic hemophiliac patients

Various immunological parameters were studied in 20 asymptomatic patients with hemophilia A, 3 patients with hemophilia B and 1 patient with von Willebrand disease. Patients were treated with cryoprecipitate or fresh frozen plasma. Significantly decreased mean percentage and absolute count (p less than 0.01) of peripheral blood E-rosette-forming cells compared to controls was found. There were normal mean percentages and absolute counts of lymphocytes, T-helper inducer, T-suppressor cytotoxic and natural killer cells. The proportion and absolute number of B cells was slightly increased. Significantly decreased natural killer cell activity (p = 0.02) of peripheral blood lymphocytes was observed. Our results indicate that asymptomatic patients with hemophilia may have early evidence of immunodeficiency.     

Immunologic response in vitro after thymechtomy in patients with myasthenia gravis.

Thymectomy in adult animals impairs immune functions such as lymphocyte response to phytohemagglutinin (PHA) and to allogeneic cells. The responses of lymphocytes from 18 myasthenia gravis patients, 12 of whom had undergone thymectomy, were studied; the interval between thymectomy and investigation ranged from 1 month to 26 years (mean, 9.5 years). Peripheral blood lymphocytes were cultured in autochthonous plasma or homologous AB serum. In vitro responses to stimulation with PHA, concanavalin A and allogeneic monomuclear cells were within the 95% range of normal responses in all patients. Because our findings contrast with the definite immune defects resulting from thymectomy found in adult animals, longer follow-up is needed.     

Immunologic status in Hodgkin patients: correlation with Epstein-Barr virus titers]

1. In Hodgkin's disease patient's immunological in vitro and in vivo parameters are of prognostic importance. 2. Skin test reactivity correlates to peripheral T-lymphocyte counts and Con A induced lymphoblastogenesis. 3. Con A is the most sensitive in vitro indicator for detecting latent immunodeficiency. 4. Hodgkin patients in long term remission after tumor reductive therapy exhibit a qualitative and quantitative lymphocyte defect. 5. In Hodgkin patients Herpes virus related antibody titers are elevated against Epstein Barr virus (EBV). The elevation coincides with a decreased T-cell number and function. Antibodies against other Herpes viruses (HSV, CMV, VZV) are in the normal range, when tested by the complement fixation method.     

Immunologic autoagression in patients with diabetic retinopathy under type I diabetes mellitus

Presence of IgG autoantibodies to broad spectrum of tissues' antigens in patients with diabetic retinopathy under type I diabetes mellitus was studied. Increased levels of antibodies to different antigens were observed in 56% of patients. Increased levels of antibodies to hypophysis, denaturated DNA, and myelin basic protein were detected most frequently. Correlation in presence of autoantibodies of different specificity with diabetes length as well as with frequency of respiratory infections was established. There was relation between level of antibodies to DNA and increased permeability of microvascular network of retina as well as between presence of autoantibodies to myelin basic protein and encephalopathy in patients with type I diabetes mellitus.     

Molecular mechanisms of systemic enzyme therapy

This work deals with the molecular mechanisms of system enzymotherapy agents action--both administrated perorally and intratestially sorbed enzymatic complexes. The basic of functional composite part of SET agents are supposed to be trypsin degraded forms.     

Anti-TNF-alpha therapy enhances the effects of enzyme replacement therapy in rats with mucopolysaccharidosis type VI

Background

Although enzyme replacement therapy (ERT) is available for several lysosomal storage disorders, the benefit of this treatment to the skeletal system is very limited. Our previous work has shown the importance of the Toll-like receptor 4/TNF-alpha inflammatory pathway in the skeletal pathology of the mucopolysaccharidoses (MPS), and we therefore undertook a study to examine the additive benefit of combining anti-TNF-alpha therapy with ERT in a rat model of MPS type VI.

Methodology/Principal Findings

MPS VI rats were treated for 8 months with Naglazyme® (recombinant human N-acetyl-galactosamine-4-sulfatase), or by a combined protocol using Naglazyme® and the rat-specific anti-TNF-alpha drug, CNTO1081. Both protocols led to markedly reduced serum levels of TNF-alpha and RANKL, although only the combined treatment reduced TNF-alpha in the articular cartilage. Analysis of cultured articular chondrocytes showed that the combination therapy also restored collagen IIA1 expression, and reduced expression of the apoptotic marker, PARP. Motor activity and mobility were improved by ERT, and these were significantly enhanced by combination treatment. Tracheal deformities in the MPS VI animals were only improved by combination therapy, and there was a modest improvement in bone length. Ceramide levels in the trachea also were markedly reduced. MicroCT analysis did not demonstrate any significant positive effects on bone microarchitecture from either treatment, nor was there histological improvement in the bone growth plates.

Conclusions/Significance

The results demonstrate that combining ERT with anti-TNF- alpha therapy improved the treatment outcome and led to significant clinical benefit. They also further validate the usefulness of TNF-alpha, RANKL and other inflammatory molecules as biomarkers for the MPS disorders. Further evaluation of this combination approach in other MPS animal models and patients is warranted.