Permeafix: a useful tool to detect antigens and DNA in flow cytometry.

A new product called Permeafix has been developed for gentle fixation of cells and permeability of their membranes. This product is useful for the staining of intracellular antigens and parallel quantification of DNA content simultaneously with cell surface staining.

Matrigel: a useful tool to study endothelial differentiation.

Matrigel is a matrix of a mouse basement membrane neoplasm. It represents a complex mixture of basement membrane proteins including laminin, type IV collagen, entactin/nitrogen and proteoheparan sulfate, but it also contains growth factors. Matrigel induces endothelial cells to differentiate as evidenced by both the morphologic changes and by the reduction in proliferation and, therefore, offers a convenient model to study biochemical and molecular events associated with angiogenesis. Further, Matrigel permits to study the roles of the extracellular matrix in angiogenesis.

The variations of the serum IgD and of the circulant immune complexes in diabetic patients.

The levels of serum IgD and of the circulating immune complexes (CIC) were determined in 168 diabetic patients, of whom 78 with type 1, 59 with type 2 and 31 with the so-called "intermediary" type of the disease, in comparison with 124 non-diabetic subjects for IgD and 100 for CIC. The results revealed very low IgD titres (less than 1 mg%, considered undetectable) in almost 3/4 of the cases; values over 1 mg% were recorded mostly in the cases of type 1, followed by those of "intermediary" and of type 2 diabetes. The mean CIC values of 67.13 +/- 36.53 optic density units (O.D.U.) were significantly higher than in the non-diabetic controls. Certain differences with respect to age, diabetes type, duration of the disease and of the insulin therapy were also recorded. The data are interpreted with caution, further investigations being necessary to the assertion of definite conclusions.

Decrease of serum immunoglobulin M levels in some diabetic patients.

Contradictory results have been published on serum immunoglobulin levels in diabetes. Our study population consisted of 26 "juvenile" IDDM patients (males/females 14/12, mean age 15.8 +/- 2.4 yrs), 42 "adult-onset" IDDM patients (25/17, 45.1 +/- 15.2 yrs), 62 NIDDM patients (27/35, 59.8 +/- 7.7 yrs), 128 controls. IgM has been measured by a highly standardized endpoint radial immunodiffusion. Since age and sex significantly influence serum IgM levels, we calculated Z values using the formula: log (Xobs:Xexp): SDexp, where Xobs is the measured IgM in any individual and Xexp and SDexp are the expected (geometric) mean and standard deviation of the log IgM values for each year of age in both sexes, as previously calculated by orthogonal polynomials from a large population of "laboratory controls" (n = 755; 10-70 yrs). These Z values (+/- SD) in juvenile IDDM (-0.442 +/- 0.988) and NIDDM (-0.559 +/- 1.215) were significantly lower (P = 0.035 and P less than 10(-3)) than in "laboratory controls" (0 +/- 1). Values in adult IDDM (-0.0225 +/- 1.213; P = 0.24) and in controls (-0.018 +/- 1.04) did not differ significantly from the "laboratory controls". The prevalence of diabetes within the four quartiles of the IgM distribution differed significantly from the one expected according to the null hypothesis (chi-square = 42.2; 3df; P less than 10(-4]. This is also applied to juvenile IDDM (P less than 0.05) and NIDDM (P less than 10(-4], but not to adult-onset IDDM. These results suggest that the low IgM levels may partly contribute to the poorly explained increase in susceptibility to infections in some diabetics.

Is microalbuminuria a different problem for type I and type II diabetes mellitus?

As in previous studies an improved single radial immunodiffusion technique was used for albumin, calculating the albumin/creatinine ratio in spot urine (mg/g/1.73 m2). This ratio was 4.7 +/- 0.0174 (geometric mean +/- SEM as logarithm) in 130 healthy controls, the highest value being 10.8. The 182 non-selected ambulatory diabetic patients presented three subgroups, each showing a non-gaussian frequency distribution: 47% with normal values (5.0 +/- 0.0224); 42% with ratio values from 11.0 to 88.8 (22.7 +/- 0.0269, significantly differing from the controls); 11% with clinical proteinuria (subsequently excluded from the study). Type I (9.6 +/- 0.0452; n = 76) and type II diabetic patients (10.7 +/- 0.0430; n = 86) significantly differed (p less than 0.001) from the controls but not from one another. Irrespective of the diabetes type, ratio values were significantly correlated with the duration of diabetes, age of patients, age of diagnosis (for instance 16.2 +/- 0.0974 in 15 patients aged greater than 65 years versus 9.1 +/- 0.0792 in 23 patients aged less than 20 years), glycemia level and chronic complications (especially retinopathy). Therefore, more than half the diabetic patients, non-selected, presented an increased albumin excretion as compared to the controls. On the other hand, microalbuminuria appears to be linked to age, duration of the disease and quality of the metabolic control rather than to the diabetes type.

The metabolic behaviour of the cellular populations in the pleural or peritoneal effusions from patients with malignant diseases.

Complex morphologic, biochemical and cyto-enzymochemical investigations have been performed in the pleural or peritoneal effusions which occurred in 80 patients with various neoplastic diseases (pleural, pulmonary, hepatic, genital or breast cancers). As shown by the morphologic and biochemical tests, the proportion of fluids with a malignant or benignant character varied widely in the different forms of neoplasias. In the same effusions, the authors have studied the metabolic behaviour of acid phosphatase activity, the glycogen and nucleolar RNA amounts in the lymphocytes, in the malignant cells and in the mesothelial ones. High values of these cyto-enzymochemical components were found in the lymphocytes and the malignant cells from fluids with a positive malignant character, although similar changes were also observed in many cells of the effusions with morphologically negative reactions, and in a lesser proportion in those from patients with adenocirrhosis. It is concluded that such investigations could contribute to a better understanding of the metabolic behaviour of the cells from neoplastic effusions thus increasing the possibility of detecting the malignant character of the cells in the pleural or peritoneal effusions with a morphologically negative character.

Correlations between insulin antibodies and the HLA system in a group of type I diabetic patients in Bucharest.

Investigations were carried out in Bucharest in 102 patients with insulin-dependent diabetes mellitus (IDDM) in order to verify the possible existence of a relationship between the HLA system and the level of insulin antibodies. The A and B loci were tested with monospecific antisera, using PEC as a separation agent for the determination of the insulin antibody titer. Group I, without antibodies (34 cases), presented: HLA-B7 (18.89% of total specificities), A3 (11.02%), A1 and B5 (8.74% each), etc.: for haplotypes the following were found: HLA-A3/B7 (9.91% of the total haplotypes), A2/B7 (8.26%), A1/B7 (5.78%). Group II (50 cases) with low or medium titers (less than 30% binding) included: HLA-B7 (20.47%), A2 (11.88%), (A1 (9.44%), B5 (8.74%), and haplotypes: HLA-A2/B7 (8.88%), A1/B7 (7.22%), A3/B7 (6.11%). Group III (18 cases), with high antibody titers (greater than 30% binding), presented: HLA-B7 (20.28%), A1 and A2 (11.59% each), A10 (10.14%), and haplotypes: HLA-A1/B7 (10.60%), A2/B7 and A10/B7 (9.09% each), A3 B7, A2 B5 and A10/B5, (6.06% each). Irrespective of the insulin antibody titer, B7 antigen surprisingly appeared predominant in our cases. Moreover, a marked tendency of A1, and to a certain extent of A2, to increase in terms of the titer was noted in parallel with a significant decrease of A3.

The metabolic behaviour of the lymphocytes from serous effusions in various benign internal diseases.

The functional status of the lymphocytes from pleural or peritoneal effusions occurring in 148 patients with various internal diseases such as tuberculosis, liver cirrhosis, heart failure and pneumonias was studied by laboratory investigations including morphologic examination, cytoenzymatic, cytochemical and biochemical tests for determining the scores of acid phosphatase activity and of endolymphocytic glycogen, as well as the presence of nucleolar RNA. These values were found increased in the fluids from tb patients and in some cases of liver cirrhosis, heart failure and metapneumonic pleurisies. The variations of these parameters are assumed to provide information on the metabolic behaviour of the lymphocytes present in these fluids and on their participation to the immune, inflammatory processes which occur in the course of some of the diseases investigated.

Study of some lymphocyte metabolic parameters in diabetes mellitus.

Cytochemical study of peripheral lymphocytes in 78 diabetic patients and 22 controls included the determination of glycogen (with calculation of the "glycogen score"), lipids and nucleolar RNA (with calculation of the so-called "lymphocyte nucleologram"). All three metabolic components showed marked differences between the diabetes and the controls. Further analysis in terms of sex, patient's age, duration of disease, basic treatment and main complications of diabetes, also showed certain variations of the above parameters but of lesser amplitude. Metabolic alterations of lymphocytes in diabetes mellitus reflect the pathogenic particularities of the disease, and might probably be used as a basis for further practical purposes.

Study of some erythrocytic enzymes in extracorpuscular hemolytic anemias.

Investigations on the activity of three erythrocytic enzymes i.e., glucose-6-phosphate dehydrogenase (G-6-PDH), catalase and lactate dehydrogenase (LDH), in 20 patients with extracorpuscular hemolytic anemias of various origins showed a general tendency to decrease of G-6-PDH and catalase, with a concomitant increase in LDH. These results are interpreted as due to metabolic disturbances induced by hemolysis in the erythroblastic series and/or to possible perturbations specific for each type of anemia.

Circadian variations of certain blood components currently investigated.

The circadian variations of serum components such as: cholesterol, LDH, GOT, alkaline phosphatase, proteins, bilirubin, ferrous ions and creatinine were investigated. Blood collection was made two times in 24 hrs.: 5-6 p.m. and 7-8 a.m. on the next day. The data obtained allowed the classification of the subjects into two groups: stable and variable. The most interesting observations were: evening decrease of cholesterol, evening increases of LDH with a tendency of intensification of the LDH5 fraction in the zymogram and evening decreases of ferrous ion.

Attempts to improve the early enzymatic diagnosis of coronary heart disease.

Based on the hypothesis that beta lipoproteins have the ability to trap certain enzymes, an attempt was made to release these enzymes from the eventual beta lipoprotein blockade, by subjecting the serum to different processings such as: freezing-thawing (3 times), ultrasonation or treatment with Triton X100 or sodium desoxycholate. After the first two procedures an increase of serum LDH and alkaline phosphatase activities was observed in about half the sera investigated (normal and pathologic). In few cases of coronary heart disease (two out of 60), LDH activity reached a clearly pathologic level.

Variations of salivary amylase as a test of internal irradiation in man.

The hypothesis of the parotid gland radiosensitivity has suggested to the authors to follow up the effect of therapeutic internal irradiation on the serum, urine and salivary alpha-amylase activity in man. The assay and visualization of alpha-amylase isoenzymes on agar gel zymograms by using the "Phadebas amylase test" tablets has demonstrated in some cases increases of salivary alpha-amylase activity expressed by the ratio of the optical density after internal irradiation to that before administration of radioisotopes.

Cytoenzymochemical effects of some antiblastic drugs and prediction of response to chemotherapy in acute leukemias.

Over 20 cytoenzymochemical tests were carried out in 152 patients with different types of acute leukemia to estimate the effects of some antiblastic drugs such as L-asparaginase, Purinethol, Methotrexate, Endoxan, Vinchristine, Cytosine Arabinoside a.o. The patients selected for the study were carefully examined before treatment at different moments during and/or at the end of the treatment. The effects of these drugs on the blast cells were mild when the cellular populations had a low rate of nucleic acid synthesis, high glycogenic score and high amounts of lipids or an important oxidative enzymatic activity. The enzymatic prediction tests: the acid phosphate deviation test and the succinic dehydrogenase inhibition test including the variant suggested by some of the authors - the latic dehydrogenase inhibition test - gave satisfactory results only in certain cases of acute leukemia.