A comparison of the kinetic properties of the common and rare variants of adenosine deaminase

Adenosine deaminase activity has been measured in red cells from individuals of known ADA phenotype (ADA 1, ADA 2-1, ADA 3-1, ADA 3-2) using adenosine and 2'-deoxyadenosine as substrates. No significant differences were observed among the phenotypes in their relative deaminase activity with the two substrates. However, evidence suggests the occurrence of an uncommon allele designated ADA ^1wdetermining low levels of ADA activity.
The deaminase activities of the phenotypes were in the order ADA 1 > ADA 2-1 > ADA 3-1 > ADA 3-2 with both substrates. The relative activities of the alleles were estimated to be: ADA ¹ 100%, ADA ² 89%, ADA ³ 28% and ADA ¹ 67% with adenosine, and ADA ¹ 100%, ADA ² 87%, ADA ³ 39% and ADA ¹ 66% with 2'-deoxyadenosine.
The Michaelis constants for adenosine and 2'-deoxyadenosine were determined for the different phenotypes. There were no significant differences in these values among the phenotypes.     

Genetic polymorphism of esterase B3 in human leukocytes

Human tissues contain an esterase activity called ESB3, detectable by starch gel electrophoresis followed by staining with α-naphthyl butyrate. Using mononuclear leukocytes, we demonstrated an electrophoretic variant of ESB3. Family studies suggest that the variant is inherited as a simple Mendelian trait; individuals with the ESB3 2-1 phenotype are heterozygotes, designated ESB3 ¹ ESB3 ², to distinguish them from the more common homozygotes, ESB3 ¹ ESB3 ¹. The frequency of the ESB3 ² allele is estimated to be 0.035 in U.S. Whites. No homozygotes for this allele have yet been found.
Our studies suggest that the enzyme from ESB3 1 individuals exists primarily as a trimer of three identical subunits with a molecular weight of approximately 58000 daltons. The genetic variant ( ESB3 ² allele) appears to be the result of a mutation that does not affect the charge of the subunit, but rather reduces its ability to form and maintain the trimeric structure.     

δ-aminolevulinate dehydrase: a new genetic polymorphism in man

A method has been developed for the electrophoretic and quantitative analyses of human red cell δ-aminolevulinate dehydrase (ALADH). The enzyme is under the control of an autosomal gene, with two common codominant alleles, ALADH ¹ and ALADH ², with frequencies of 0–89 and Oil, respectively, in the Italian population. Mean phenotypic enzyme activities are nearly identical: 52, 49 and 55 mlU/g Hb for ALADH 1, 2-1 and 2 phenotypes respectively.     

The biochemical genetics of human γ-aminobutyric acid transaminase

1. Two methods have been devised for the detection after electrophoresis of γ-aminobutyric acid transaminase (GABAT) isozymes.
2. GABAT isozymes can be detected in liver, brain, kidney, pancreas, heart, testis, spinal cord and upper jejunum. The greatest activity occurs in liver.
3. Three different commonly occurring electrophoretic types of GABAT have been identified. It seems likely that they are determined by two alleles at an autosomal locus (GABAT).
4. The gene frequencies of GABAT ¹ and GABAT ² in a random sample of European livers were 0.56 and 0.44 respectively.
5. The three banded patterns seen in heterozygotes suggest that GABAT is a dimeric enzyme.
6. GABA, β-alanine and 5-aminovaleric acid can act as substrates for GABAT.
7. GABAT activity can be demonstrated in all areas of human brain with the exception of the corpus callosum. Brain samples from patients with Huntington's chorea show no abnormal GABAT activity or unusual phenotypes.     

Immunohistochemical characterization of thymic macrophages in normal and treated rats: a differential sensitivity to cyclosporine A

In the present study, a set of two monoclonal antibodies (TRPM1, TRPM2) was used to investigate the macrophage populations in the rat thymus and their different sensitivities to cyclosporine-A (CsA). With double immunohistochemical staining we demonstrated that, in the normal rat thymus, there are 3 populations of macrophages (TRPM1⁺, TRPM1/2⁺, TRPM2⁺), present in different proportions throughout the thymus. In the outer cortex TRPM1⁺ and TRPM1/2⁺ were present, but the TRPM1/2⁺ cells were more numerous. No TRPM2⁺ cells were observed in this area. The cortex and medulla showed all 3 types of cells with a majority of TRPM1/2⁺ cells. In the corticomedullary zone (CMZ) TRPM1/2⁺ and TRPM2⁺ macrophages were present in about equal proportion while only a few TRPM1⁺ cells were observed. After CsA treatment (for 21 days) profound changes occurred in the thymus; we observed a complete disappearance of the thymic medulla and a reduction in the total number of macrophages. The TRPM1⁺ macrophages had been eliminated, a few TRPM1/2⁺ cells were found while many of the cells were TRPM2⁺. The presence of the macrophages in different thymic areas suggests that they are a very heterogeneous population. The possible significance of the macrophage heterogeneity and the relationship to CsA sensitivity is discussed.     

Genetic polymorphisms of the Pmol and Pmo2 salivary proteins detected by the modified protein staining method

Two polymorphic proteins, Pmo1 and Pmo2, were found in human parotid saliva by modifying the protein staining method of Sung & Smithies (1969). The inheritance of each polymorphism was controlled by a dominant allele at an autosomal locus. This hypothesis was supported by studies in 50 families including 103 children. The gene frequencies were Pmo1 ⁺= 0.308, Pmo1 ^-= 0.692, Pmo2 ⁺= 0.026, Pmo2 ^-= 0.974. The Pmo1 and Pmo2 proteins reacted immunologically with antisera prepared to salivary proline-rich proteins (Pr and Gl). The isoelectric point was in excess of 8.58. These results showed that the Pmo1 and Pmo2 proteins belong to the basic proline-rich proteins in human parotid saliva.     

Cytokine and chemokine expression in the skin from patients with maculopapular exanthema to drugs

Background:  Maculopapular exanthema (MPE) is the most frequent clinical manifestation of nonimmediate allergic reactions to drugs and T helper 1 (Th1) cytokines and CD4⁺ T cells have been shown to play an important role in its pathogenesis. We assessed the role of cytokines and chemokines and their receptors in the pathogenesis of MPE.
Methods:  We evaluated skin biopsies and peripheral CD4⁺ and CD8⁺ T cells from 27 patients during the acute phase of the reaction and 26 exposed controls. Semiquantitative real-time PCR was performed to determine the expression of cytokines and chemokines and their receptors and immunohistochemistry was used to determine the same chemokines and their receptor proteins in skin.
Results:  There was a high expression of the Th1 cytokines interferon-γ ( P  = 0.006) and tumor necrosis factor-α ( P  = 0.022) in skin and CD4⁺ T cells ( P  = 0.007 and P  = 0.005, respectively); and of the Th1 chemokines CXCL9 ( P  = 0.005) and CXCL10 ( P  = 0.028) in the skin, while their receptor CXCR3 was increased in skin ( P  = 0.006) and CD4⁺ T cells ( P  = 0.03). Homing chemokine receptors were also increased: CCR6 in skin ( P  = 0.026) and CD4⁺ T cells ( P  = 0.016), and CCR10 only in CD4⁺ T cells ( P  = 0.016), as well as their ligands, CCL20 and CCL27, in skin alone. Immunohistochemistry confirmed these results.
Conclusions:  These data show significant differences in the expression of chemokines and chemokine receptors, related with a Th1 profile, in both skin biopsies and peripheral CD4⁺ T cells in patients with drug-induced MPE.     

Inheritance of Gm(g) and a gene complex GmaGmg weak

Gm(a) (x) (g) (f) (b¹) were examined in 84 random Norwegian families with 444 children allowing direct estimates of the f,b, a,g, ax,g , and a,b (including a,b ⁰ b ¹ , a,b ⁰ st , and f,b ⁰ b ¹) gene complex frequencies and an upper estimate of the f,g frequency. Phcnotype and mating type patterns were random (non–assortative).
Disturbed segregation ratios were found in ax,g/f,b×f,b/f,b families irrespective of sex of segregating parents, and in families with a,g/f,b in backcross if segregating parents were separated by sex. In the latter case excess a,g from females was balanced by excess f,b from males, this trend being present also in two other Norwegian family materials (tested for Gm(a) (x) (b¹)).
Gm(n) was examined in part of the material, allowing approximate frequency estimates of the polymorphic f,b,n, f,b,n–, a,g,n– , and ax,g,n– and the idiomorphic ax,g,n, a,g,n, a,b ⁰ st,n– and a,b b ¹,n– ( fa, b ⁰ b ¹ unassigned). The information on the IgGl–IgG3 and the IgGl, IgG3–IgG2 linkage relations were estimated.
A family with familial low Gm(g) activity was observed and interpretations included a genetically linked cis–IgC3–suppressor gene in analogy with thalassemia genes as well as a structural allele mutation.     

Studies of S-adenosylhomocysteine-hydrolase polymorphism in a Croatian population

Recently, a proven case of human S-adenosylhomocysteine-hydrolase (SAHH) deficiency was reported in a Croatian boy. As molecular analysis of the SAHH gene in this case revealed two different mutant alleles, we investigated the polymorphism of human SAHH in a total of 237 red blood samples from unrelated Croats using starch gel electrophoresis and an enzyme-specific staining procedure. From the relative enzymatic activity of SAHH—determined by densitometric assessment of electrophoretic patterns, and calculated on the basis of the protein concentration of the red blood cells—we detected three individuals as being heterozygous for an SAHH 0-allele. Moreover, a total of four different electromorphic SAHHs have been observed, giving allele frequencies calculated as SAHH 1=0.941, SAHH 2=0.032, SAHH 3=0.006, SAHH 4=0.015, and SAHH 0=0.006.     

Intestinal colonization of a human subject by vancomycin-resistant Enterococcus faecium

Objective: To study the ability of two strains of vancomycin-resistant Enterococcus faecium to colonize the human intestine.
Methods: A single human subject ingested separately two strains of vancomycin-resistant E. faecium isolated from a pig and a chicken. The feces were cultured on selective medium. Prior to ingestion no vancomycin-resistant cocci were present in the feces. Ingestion of 10⁴-10⁵ CFU resulted in either no colonization or isolation only after enrichment. Ingestion of 10⁷ CFU of one strain resulted in colonization for a period of nearly 3 weeks, with fecal counts at times in excess of 10⁶ CFU/g. Ingestion of similar numbers of the other strain and reingestion of the first strain resulted in excretion in the feces for much shorter periods. When the fecal count of the ingested strains was greater than 10⁴-10⁵ CFU/g, the strains were isolated from swabs taken from perianal skin.
Conclusions: Vancomycin-resistant E. faecium strains from pigs and poultry are able to colonize the human gut and the perianal skin.     

Determination of in vitro postantibiotic effects in Staphylococcus aureus and Escherichia coli by [3H]thymidine incorporation

Postantibiotic effects (PAE) and control-related effective regrowth time (CERT) of dicloxacillin, vancomycin, rifampin and gentamicin in Staphylococcus aureus and imipenem, gentamicin, tobramycin, doxycycline and rifampin in Escherichia coli were measured by standard viability counting and [³H]thymidine incorporation. For PAE determination, the two methods correlated well; r ² = 0.821 for S. aureus and r ² = 0.939 for E. coli . For viable counts below the detection limits of 10⁵ to 10⁶ log₁₀ CFU/mL, the PAE was overestimated by the [³H]thymidine method. Quantitation of CERT by both methods showed a good correlation, r ² = 0.867 for S. aureus and r ² = 0.997 for E. coli . Measuring [³H]thymidine incorporation in bacteria is a novel alternative method for the determination of PAE and CERT.     

New variants of Ps salivary polymorphic proteins

Electrophoretic analysis of the Ps protein demonstrated the existence of phenotypes additional to those described by Azen & Denniston (1980). A hypothesis that the polymorphism of the Ps protein is determined by five expressed and one unexpressed alleles was supported by family studies. The gene frequencies in a Japanese population were Ps ^1F= 0.0016, Ps ¹= 0.2983, Ps ^2F= 0.0288, Ps ^2S= 0.0079, Ps ³= 0.0111, Ps ⁰= 0.6523.     

Effects of Peritoneal Fluid from Endometriosis Patients on Interferon-γ-Induced Protein-10 (CXCL10) and Interleukin-8 (CXCL8) Released by Neutrophils and CD4+ T Cells

Problem  Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-γ-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4⁺ T cells, and monocytes.
Method of study  Neutrophils, CD4⁺ T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay.
Results  The addition of ePF to cultures of CD4⁺ T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF ( R  = 0.89, P  =   0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4⁺ T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils ( R  = 0.89, P  <   0.001), CD4⁺ T cells ( R  = 0.93, P  <   0.001), and monocytes ( R  = 0.70, P  =   0.01). Moreover, the addition of ePF significantly enhanced the interferon-γ-induced release of IP-10 by nuetrophils and CD4⁺ T cells.
Conclusion  These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.     

Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow

Problem  Macrophages are recruited in large number to the interplacentomal endometrium of the cow during pregnancy. We evaluated whether endometrial macrophages also accumulate in placentomal regions of endometrium during pregnancy and whether endometrial macrophages are regionally differentiated.
Method of study  Interplacentomal endometrium and placentomes were subjected to dual-color immunofluorescence using CD68 as a pan-macrophage marker.
Results  CD68⁺ cells were abundant in stroma of the interplacentomal endometrium and caruncular septa of the placentomes. CD68⁺ cells were not present in fetal villi of the placentomes or in the interplacentomal chorion. Regardless of location, the majority of CD68⁺ cells also expressed CD14. In interplacentomal endometrium, CD68⁺CD11b⁺ cells were present in deeper areas of the stroma but not in shallow endometrial stroma. In caruncular septa of the placentome, CD68⁺ cells were negative for CD11b. CD68⁺ cells in the interplacentomal endometrium were negative for MHC class II while most CD68⁺ cells in caruncular septa were positive for MHC class II.
Conclusion  CD68⁺CD14⁺ macrophages present in the stroma of the interplacentomal endometrium and caruncular septa of the placentome are regionally differentiated with regard to expression of CD11b and MHC class II.     

Peripheral T-cell Lymphomas Immunologic and Enzyme Histochemical Analysis of 15 Cases

Ffteen cases of peripheral T cell lymphoma were studied to evaluate the respective properties of various histologic types using enzyme histochemical and ultrastructural examinations in addition to immunological methods. Eleven cases in an ATLA negative group manifested various histologic patterns such as IBL like, pleomorphic and Lennert's lymphomas in comparison with the relatively monomorphic proliferation of neoplastic lymphoid cells in the 4 ATLA positive cases. The presence of neoplastic clear cells is characteristic of peripheral T-cell malignancies, and is likely to be found in CD4 lymphomas. There is an occasional reaction of epithelioid histiocytes and plasma cells with eosinophils, the former being designated Lennert's lymphoma and the latter IBL like T-cell lymphoma. Immunological examination revealed four immunophenotypic patterns: (1) CD2⁺3⁺4⁺8⁺, (2) CD2⁺ 3⁻4⁺8⁻, (3) CD2⁺3⁺4⁻8⁺, and (4) CD2⁺3⁺4⁺8⁺, but did not provide information concerning the intimate relationship between histologic types and immuno phenotyes. β-Glucuronidase reactivity, however, contributed to the distinction between helper and suppressor T cell malignancies, suggesting its usefulness for distinguishing these two cell types and their malignant counterparts.     

Effect of clindamycin in a model of acute murine toxoplasmosis

Objective: To characterize the antitoxoplasma activity of clindamycin in a murine model of acute toxoplasmosis.
Method: Rates of survival and mean survival times of Swiss Webster mice infected intraperitoneally with 10⁶-10² tachyzoites of the RH strain of Toxoplasma gondii treated with clindamycin or sulfamethoxazole (positive control) or untreated (negative control) were compared. Survivors were submitted to examination of untreated brain tissue preparations, intraperitoneal and peroral subinoculations of brain tissue homogenates into fresh mice, and to patho-histology, including immunohistochemistry, of brain and lungs.
Results: The effect of clindamycin treatment (400 mg/kg/day) on infected Swiss Webster mice was inoculum size dependent, ranging from no survivals in animals infected with 10⁶ parasites, to 100% survivals with an inoculum of 10². Treatment initiated 24 h before and at time of infection prolonged mean survival times comparably to sulfamethoxazole, and significantly when compared to untreated controls. In contrast, treatment initiated 48 h postinfection with an inoculum of 10⁶ did not postpone death. In the clindamycin-treated survivors, there was no biological or histologic evidence for the persistence of toxoplasma.
Conclusions: The results obtained show that at an appropriate parasite dose/drug dose ratio, clindamycin is strongly toxoplasmacidal in a murine model of acute toxoplasmosis.     

Association of HincII RFLP of low density lipoprotein receptor gene with obesity in essential hypertensives

Obese (BMI 26 kg/m²; n=51) and lean (BMI <26 kg/m²; n=61) Caucasian patients with severe, familial essential hypertension, were compared with respect to genotype and allele frequencies of a Hin cII RFLP of the low density lipoprotein receptor gene ( LDLR ). A similar analysis was performed in obese (n=28) and lean (n=68) nonmotensives. A significant association of the C allele of the T → C variant responsible for this RFLP was seen with obesity ( x ²=4.6, P =0.029) in the hypertensive, but not in the normotensive, group (odds ratio=3.0 for the CC genotype and 2.7 for CT ). Furthermore, BMI tracked with genotypes of this allele in the hypertensives ( P =0.046). No significant genotypic relationship was apparent for plasma lipids. Significant linkage disequilibrium was, moreover, noted between the Hin cII RFLP and an Apa LI RFLP ( x ²=33, P <0.0005) that has previously shown even stronger association with obesity (odds ratio 19.6 for cases homozygous for the susceptibility allele and 15.2 for heterozygotes). The present study therefore adds to our previous evidence implicating LDLR as a locus for obesity in patients with essential hypertension.     

ASRI2005-88 Comparable levels of CD4+CD25+ CTLA4+ Treg cells in peripheral blood of pre-eclampsia and normal pregnant patients

The acceptance of the semiallogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, T-regulatory cells were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Tregulatory cells (Treg). The major aim of the present work was to investigate if pre-eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks. Therefore, we measured the surface antigens CD4, CD25, CD8, CTLA4 (as well as the secretion of IL-10) in peripheral blood from patients suffering from pre-eclampsia (n = 8) and age-matched patients undergoing normal pregnancies (n = 9) by 4-colour flow-cytometry. We were not able to find any significant differences in the levels of CD4⁺, CD25⁺, CD8⁺, CTLA4, CD4⁺/CD25⁺, CD4⁺/CD25^bright, CD4⁺/CTLA4, CD25⁺/CTLA4, CD4⁺/CD25⁺/CTLA4, CD8⁺/CD25⁺, CD8⁺/CTLA4 or CD8⁺/CD25⁺/CTLA4 cell subsets. Our data suggest that Treg may not participate in the onset of pre-eclampsia and suggest other regulatory mechanisms during late pregnancy.