Innovative therapeutic approach: sequential treatment with plasma exchange and eculizumab in a pregnant woman affected by atypical hemolytic-uremic syndrome.

The atypical HUS (aHUS) is a rare genetic disease, with poor prognosis, characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. This syndrome is often related to mutations in the genes encoding complement regulatory proteins. A 26-year-old woman with homozygous mutation in complement factor H (CFH) developed a relapse of aHUS at 17th week of pregnancy. Despite treatment with plasma exchange (PEX), at the 26th week of gestation eculizumab was started. The sequential treatment with eculizumab after PEX was well tolerated and it has led to clinical remission.

Enhancement of lysosomal glycohydrolase activity in human primary B lymphocytes during spontaneous apoptosis.

It has been shown that lysosomes are involved in B cell apoptosis but lysosomal glycohydrolases have never been investigated during this event. In this study we determined the enzymatic activities of some lysosomal glycohydrolases in human tonsil B lymphocytes (TBL) undergoing in vitro spontaneous apoptosis. Fluorimetric methods were used to evaluate the activities of beta-hexosaminidases, alpha-mannosidase, beta-mannosidase, alpha-galactosidase, beta-glucuronidase and alpha-fucosidase. Results show that in TBL during spontaneous apoptosis, there is a significant increase in the activity of beta-hexosaminidases, alpha-mannosidase, beta-mannosidase and beta-galactosidase. Also beta-glucuronidase and alpha-fucosidase activities increase but not in a significant manner. Further studies on beta-hexosaminidases revealed that also mRNA expression of the alpha- and beta-subunits, which constitute these enzymes, increases during spontaneous TBL apoptosis. When TBL are protected from apoptosis by the thiol molecule N-acetyl-L-cysteine (NAC), there is no longer any increase in glycohydrolase activities and mRNA expression of beta-hexosaminidase alpha- and beta-subunits. This study demonstrates for the first time that the activities and expression of some lysosomal glycohydrolases are enhanced in TBL during spontaneous apoptosis and that these increases are prevented when TBL apoptosis is inhibited.

Early detection of insulin deprivation in continuous subcutaneous insulin infusion-treated patients with type 1 diabetes.

BACKGROUND: This study was performed to define the clinical relevance of early changes of capillary 3beta-hydroxybutyrate (3betaOHB), for detection of metabolic deterioration before occurrence of overt diabetic ketoacidosis following interruption of continuous subcutaneous insulin infusion (CSII). METHODS: An open clinical trial was performed with eight patients with type 1 diabetes on CSII therapy. After an overnight fast, at 8 a.m. (T0) CSII was interrupted for 4 h. At noon (T240) CSII was re-established, and at 4 p.m. (T480) the study was ended. Blood glucose (BG) and capillary and plasma 3betaOHB were measured at 30-min intervals, plasma insulin at 60-min intervals, and urinary ketones at 120-min intervals. RESULTS: After CSII interruption mean BG increased from 149.8+/-54.4 mg/dL at T0 to 224.8+/-56.2 mg/dL at T240 (P<0.05), and mean capillary 3betaOHB increased from 0.1+/-0.1 mmol/L at T0 to 0.9+/-0.6 mmol/L at T240 (P<0.001). The rate of increase of capillary 3betaOHB was faster and significantly more relevant than that of BG (P<0.05). The restoration of CSII produced a significant reduction of mean BG and capillary 3betaOHB (T480, 119.5+/-24 mg/dL and 0.2+/-0.2 mmol/L, respectively; P<0.05 for both vs. T240). The recovery of capillary 3betaOHB was significantly faster than that of BG (P=0.03). CONCLUSIONS: The dynamic evaluation of changes of capillary 3betaOHB levels can represent a useful support to home BG monitoring in the event of CSII interruption, providing faster information on early metabolic deterioration due to insulin deprivation and allowing preventative action for avoiding the evolution towards overt diabetic ketoacidosis. After reintroduction of insulin infusion the monitoring of the faster recovery of 3betaOHB relative to BG can provide useful information for the prevention of late hypoglycemia due to insulin overinfusion.

In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody.

Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6. The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration. Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately. In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay. Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment. This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation.

Short- and long-term haematological surveillance of healthy donors of allogeneic peripheral haematopoietic progenitors mobilized with G-CSF: a single institution prospective study.

Healthy allogeneic donors, who were treated with G-CSF and underwent peripheral blood haematopoietic precursor collection at our Institution, were enrolled in a short- and long-term haematological surveillance protocol for a 5--7--year period. To date, 94 donors have been assessed with a mean follow-up of 30 months (4--84); for 30 subjects, the follow-up is >or=48 months. During G-CSF administration, 23/94 donors showed a significant platelet count decrease from the baseline. Pre-apheresis platelet decrement correlated with the total G-CSF dose administered, baseline platelet level and donor age. Normal platelet counts returned within 4--8 months. PMN and/or lymphocyte lower values were observed in 55/94 donors 2 weeks after G-CSF administration, with mean drops from the baseline of 40 and 36% for PMN and lymphocytes, respectively. The PMN decrease correlated inversely with donor age, as younger donors were more affected than older ones, whereas the lymphocyte decrease correlated directly with the total blood volumes processed in the apheresis courses, in particular for donors subjected to large volume leukaphereses. Long-term observation showed moderate neutrophil reduction (25% count drop from the baseline) in four of the 30 donors observed for four years or more. 14 donors showed persistent, slight lymphocytopenia (mean drop of 13%) until the third year, with recovery in the fourth year of follow-up.

Maturation of the regulation of growth hormone secretion in young males with hypogonadotropic hypogonadism pharmacologically exposed to progressive increments in serum testosterone.

To study the onset of the action of gonadal sex steroids on the GH axis in spontaneous puberty, which is prolonged and sparingly predictable, we present a clinical investigative paradigm in which six previously untreated boys with isolated hypogonadotropic hypogonadism were exposed to progressively higher testosterone levels designed to mimic the androgen environment recognized during the early stages of puberty. We administered three incremental doses of testosterone (25-, 50-, and 100-mg im injections), each over a period of 4 weeks. Studies of overnight pulsatile GH secretion and GH responses to GHRH alone or combined with L-arginine (a functional somatostatin antagonist) were performed before testosterone administration and after each dose of testosterone. Serum testosterone, but not estrogen, levels increased progressively in all subjects during therapy. Deconvolution analysis of GH release profiles disclosed that GH secretory burst mass was stimulated significantly even by 25 mg testosterone. This parameter was not altered further by higher doses of testosterone. Spontaneous GH secretory burst number and amplitude increased significantly only after the 50- and 100-mg testosterone treatments, after which the serum GH response to GHRH and arginine also rose significantly. In contrast, the GH response to GHRH alone was not significantly affected by any dose of testosterone. Serum testosterone levels correlated significantly with the primary parameters of nocturnal GH secretion. In summary, our experimental model suggests that in males even very small increases in circulating testosterone occurring during the earliest stages of puberty are able to amplify pulsatile GH secretion. Our concomitant secretagogue data further suggest that testosterone exerts its action at different sites in the hypothalamo-somatotropic axis, i.e. directly at the pituitary level, and also at hypothalamic loci, possibly increasing both GHRH and somatostatin release.

In situ staining of bromodeoxyuridine positive cells in normal and neoplastic colony-forming units grown on plasma clots.

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In this paper a method for the detection of the labeling index of normal and neoplastic colony-forming units (CFU) growing in plasma clot semisolid medium is described and preliminary results on the cell cycle of 7th and 14th CFU granulocyte-macrophage are discussed.

Normal myeloid progenitors (CFU-GM) in multiple myeloma: a preliminary study in view of autologous BMT.

Normal granulocyte-macrophage precursors (CFU-GM) were studied in 65 multiple myeloma patients by means of culture assays. The patients were divided into separate groups on the basis of previous therapy (i.e. analysis performed at diagnosis or after chemotherapy), time elapsed from the last therapy (i.e. more or less than 1 month) and clinical features of the disease (i.e. tumor stage, immunoglobulin type, bone marrow plasma cell infiltration). The results were evaluated by Wilcoxon rank sum test and linear regression analysis. There was no statistical difference in CFU-GM cloning efficiency or in the number of CFU-GM/ml of bone marrow, even though a larger CFU-GM recovery was found in patients evaluated at diagnosis or at least 1 month or more from previous chemotherapy. In addition, no correlation was demonstrated between bone marrow plasma cell percentage and CFU-GM cloning efficiency. This finding was confirmed by the number of myeloid bone marrow cells in S-phase, assessed by the bromodeoxyuridine labeling index, which showed similar results in patients with different degrees of plasma cell infiltration. In conclusion our data indicate that the granular-monocytic lineage keeps its cell-line potentiality regardless of the degree of marrow plasma cell infiltration and the type of therapeutic approach. These data suggest that autologous bone marrow transplantation might be feasible even in patients with a large neoplastic infiltration.

Effect of pyridostigmine on the growth hormone response to growth hormone-releasing hormone in lean and obese type II Diabetic patients.

A suppressed growth hormone (GH) response to GH-releasing hormone (GHRH) in both lean and overweight type II diabetics has been reported. Pyridostigmine (PD), an acetylcholinesterase inhibitor, elicits GH secretion when administered alone and enhances the GH response to GHRH in normal subjects. The aim of our study was to evaluate the effect of PD on GHRH-stimulated GH secretion in both lean and obese type II diabetic patients. We studied 16 patients with type II diabetes mellitus (seven lean and nine obese). Eleven nondiabetic subjects (six lean and five obese) served as controls. Each subjects underwent treatment with (1) 120 mg PD orally or (2) 2 tablets of placebo orally, 60 minutes before intravenous (IV) injection of 100 micrograms GHRH-(1-29)NH2. We have found no significant differences in GH responses to GHRH between obese diabetics and obese controls. On the other hand, the absolute GH levels were significantly suppressed in lean type II diabetics compared with lean controls at 15 and 30 minutes after GHRH injection. Obese diabetic subjects had slightly but not significantly decreased GH responses to GHRH+PD compared with obese nondiabetic subjects (8.36 +/- 1.62 v 14.4 +/- 7.62 micrograms/L). Lean type II diabetics showed a blunted GH release after GHRH+PD compared with normal-weight healthy subjects (GH peaks, 15.77 +/- 2.17 v 40.88 +/- 6.17 micrograms/L, P < .05). PD enhanced significantly the GH response to GHRH in obese diabetics, obese controls, and non-obese controls (P < .05), but not in non-obese type II diabetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Parasite-specific antibody response in Trichinella sp. 3 human infection: a one year follow-up.

Sera from patients with confirmed or suspected trichinellosis were examined for 1 year to detect the presence of parasite-specific antibodies (IgG, IgM, and IgE) using the enzyme-linked immunosorbent assay (ELISA). The indirect ELISA was used to detect specific IgG (ELISA-IgG) and specific IgM (ELISA-IgM); an amplified technique proved the most reliable for detection of specific IgE (a-ELISA-IgE). The immunofluorescence (IF) test was used to detect specific IgG (IF-IgG). The patients were from an outbreak of trichinellosis in Salsomaggiore (northern Italy) in 1986. The parasite was isolated and isoenzymatically typed as Trichinella sp. 3. The specificity of our tests was greater than 95%. During the 1st period of infection, all tests used gave practically the same positivity rate (78.2-86.9%). One year after infection, ELISA-IgG gave the highest positivity rate (55%). With the other tests, the positivity rate was 20-38.5%. At the 2nd month of infection, the IF-IgG test was the most discriminating in patients with confirmed and suspected trichinellosis, but ELISA-IgG proved the most reliable test for detecting specific immunoglobulins in late human trichinellosis infection.

Phase II study of a new alkylating agent (PTT-119) in resistant-relapsed non-Hodgkin's lymphomas.

In a phase II study we evaluated the effect and toxicity of a new alkylating agent, PTT-119, in 26 patients with non-Hodgkin's lymphomas (NHL) resistant to or relapsed after other chemotherapy. PTT was scheduled by escalating the dose from 2.0 to 3.3 mg/kg every 3 weeks. Among 21 evaluable patients with NHL, 12 (57%) showed a good response (CR + PR) to PTT-119. Tolerance was acceptably good; no major side effects related to liver, cardiac, or renal toxicity were recorded. The most commonly recorded side effects were nausea and vomiting, alopecia, and phlebitis; diarrhea and drug-related fever were rarely seen. This report indicates a potential usefulness for PTT-119, a non-cross-resistant alkylating agent, in the treatment of NHL.

Comparison of the DNA Content, Bromodeoxyuridine Incorporation and Ki-67 Antigen Expression in Human Acute Myeloid Leukemia.

The cell kinetics of twenty-two acute myeloid leukemias (AML) were investigated by means of flow cytometry evaluating the S-phase DNA content, bromodeoxyuridine labelling index (BrdUrd L.I.) and Ki-67 antigen expression. Eight patients showed a good correlation between the DNA content and BrdUrd L.I., while nine gave rise to divergent results. In the remaining five patients the S-phase DNA content could not be evaluated due to the presence of an additional aneuploid population. The Ki-67 antigen expression defined the extent of the growth fraction in all cases and allowed for better characterization of the cell cycle. These results suggest that the three methods explore only partly overlapping events; thus, it seems that a reliable picture of the cell kinetics in leukemic populations can only be achieved by combining all these methods.

Beta-N-acetylhexosaminidase in the urine, kidney and serum of bromobenzene-intoxicated mice.

beta-N-Acetylhexosaminidase isoenzymes were separated from the kidney, serum and urine of normal mice and mice intoxicated with bromobenzene, using DEAE-cellulose chromatography. Both mouse serum and urine showed hexosaminidase profiles similar to the human counterparts with the presence of B (basic), I (intermediate) and A (acidic) isoenzymes. A notable feature was the presence of a high proportion of an intermediate form in mouse urine which is not always present in human urine. Hexosaminidase activity increased significantly in urine of mice intoxicated with bromobenzene. Its increase was time-dependent and due to kidney damage with a release in the urine of hexosaminidase A, I and, in higher proportion, B. No significant differences were observed in mouse kidney and serum profiles following intoxication with bromobenzene. The total activity of hexosaminidase, using 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as substrate, did not increase in the serum of mice intoxicated with bromobenzene. Both hexosaminidase activity and the isoenzyme pattern in urine can be used as indicators of kidney damage by bromobenzene intoxication.

Expression modes of urinary N-acetyl-beta-D-glucosaminidase in patients with chronic renal insufficiency.

BACKGROUND: Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity has emerged as potentially useful early marker of renal tubular injury. This activity is usually evaluated in random urine samples and is related to urinary creatinine concentration. Reports about the lack of correlation between NAG activity of 24-h urines and activity of random urine samples in some clinical and experimental situations led us to study the correlation existing between different procedures for expressing urinary NAG in patients with chronic renal insufficiency. METHODS: Thirty samples of 24-h urine and 30 random urine samples from chronic renal insufficiency patients were collected. The activity of urinary NAG was examined fluorimetrically. RESULTS: The following correlations were observed: (1) r = 0.431 (P = 0.017) for activity in random urine samples and total activity in 24-h urines); (2) r = 0.281 (P = 0.005) for activity in random samples and activity, expressed as U/l, in 24-h urines. CONCLUSIONS: The data show that collection of urine excreted over the whole day and evaluation of total daily excretion of NAG seems the method of choice, at least for patients with chronic renal insufficiency.

Lysosomal hydrolases in serum from human immunodeficiency virus-infected patients.

beta-Hexosaminidase isoenzymes were separated by DEAE-cellulose chromatography in the serum of 23 patients infected with human immunodeficiency virus at different stage of the disease. Forms corresponding to hexosaminidase B, I and A were present in pathological sera. There is an increase in the percentage of hexosaminidase I in pathological sera, that could be used as an additional marker to monitor the clinical stage of the disease. Furthermore, total activities of some lysosomal enzymes were determined in these sera. Activities of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, alpha-mannosidase and beta-mannosidase were significantly higher in the serum of patients at the C3 stage of disease than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate substrate, beta-glucuronidase and beta-galactosidase.

Elevated beta-N-acetylhexosaminidase activity in focal dystonia fibroblasts.

Specific activities of beta-D-hexosaminidase, alpha-D-mannosidase, beta-D-galactosidase and beta-D-glucuronidase were determined in fibroblasts of patients with writer's cramp and torticollis. These diseases show degenerative neurological disorders similar to those observed in lysosomal diseases. Hexosaminidase specific activities, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside and 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrates, were significantly higher in the fibroblasts of patients than in controls. No significant differences were observed in the specific activities of the other lysosomal enzymes. The increased hexosaminidase specific activities in torticollis and writer's cramp may be additional markers for these diseases.

Effect of antiorthostatic hypokinetic/hypodynamia on urinary endothelin-1 and N-acetyl-beta-D-glucosaminidase excretion in rats.

In an experimental model with rats in head-down suspension, plasma levels and urinary excretion of endothelin-1 (ET-1) and urinary excretion of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) were determined. Significant variations in time in the effective plasma ET-1 levels in the superior and inferior cava vessel blood of animals maintained for 6 days in hypogravity with respect to controls were observed. We not only found a transient increase in urinary NAG activity but also that the levels of U-ET-1 increased during head-down suspension. The simultaneous evaluation at urinary level of these two parameters could be an indication that there are different sites of renal parenchymal involvement or injury during antiorthostatic hypokinesis.

Activity and isoenzyme profile of N-acetyl-beta-D-glucosaminidase in urine from workers exposed to cadmium.

The urinary excretion of N-acetyl-beta-D-glucosaminidase (U-NAG) and urinary Cadmium (U-Cd) concentration, a measure of the metal load in the body, were evaluated in 28 workers exposed to Cd, to determine the relation between the two parameters. In urine from 22 exposed workers with U-Cd<2 microg/g creatinine (Cr) there was no significant difference in U-NAG value (0.98+/-0.59 U/gCr) compared to non-exposed (0.73+/-0.48 U/gCr). In the six workers with 2 microg/gCr < or =U-Cd<10 microg/gCr the U-NAG (2.32+/-0.61 U/gCr) was statistically (P<0.05) higher than in other workers. In both the U-Cd intervals examined there were no altered values of beta2-microglobulin from urine of exposed workers compared to non-exposed (<0.30 mg/l). The U-NAG isoenzymes were separated by DEAE-cellulose chromatography from urine of non-exposed subjects and exposed workers. The U-NAG isoenzyme profile in urine of non-exposed subjects showed a high percentage (about 95%) of the A (acid) form, a much lower percentage (about 4.5%) of B (basic) form and a negligible percentage (about 0.5%) of I (intermediate) form. In the urine of 22 exposed workers with U-Cd<2 microg/gCr, the percentages of U-NAG isoenzymes were not different from non-exposed. In the urine of six workers with 2 microg/gCr< or =U-Cd<10 microg/gCr the percentage (8.34+/-0.91) of isoenzyme B (U-NAG-B), a marker of lesional enzymuria, was statistically increased (P<0.05) compared to non-exposed (4.42+/-0.56). These results suggest that adopting a biological limit for U-Cd equal to 10 microg/gCr might not be sufficiently protective. The increase in U-NAG-B content at 2 microg/gCr

N-acetyl-beta-D-glucosaminidase activity in urine of dental personnel.

BACKGROUND: Dental personnel is exposed to several potential nephrotoxic agents. Urinary N-acetyl-beta-d-glucosaminidase (U-NAG) activity has emerged as a sensitive marker of early nephrotoxicity. METHODS: U-NAG was evaluated, by fluorimetric assay, in urine from 30 healthy subjects and 30 dental personnels. RESULTS: The median value of U-NAG activity (133.5 U/mmol urinary creatinine (U-Cr) in urines of dental personnel was not statistically different (P>0.05) from activity (100.7 U/mmol U-Cr) of control urines. CONCLUSIONS: The results suggest that, for dental personnel, exposure to potential nephrotoxic agents is not usually high enough to increase U-NAG activity.

beta-hexosaminidase, alpha-D-mannosidase, and beta-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I.

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.