Biomedical subjects
Publications and source records attributed to P Ruiz.
Five patients with non-urinary tract infections caused by Corynebacterium urealyticum were seen during an 8-year period at a university hospital in Madrid. Bacteremia (one case) and wound infections (four cases) were the most relevant features of these cases. Treatment with vancomycin, surgery, and supportive measures contributed to a favorable outcome for four of the five patients.
Induction of neonatal H-2 tolerance is highly efficient in most allodisparate strain combinations, with the exception of those in which neonatal recipients do not, but donor cells do, express I-E molecules. For example, less than 25% of I-E-nonexpressing B10.S mice become tolerant following neonatal injection with I-E+ (B10.AxB10.S)F1 cells. It has been hypothesized that the high rate of nontolerance observed in I-E-nonexpressing neonatal recipients of injections of I-E-bearing cells is caused by a failure to delete V beta 11+ cells in the immediate postnatal period. To test this possibility, we have examined V beta 11 expression and tolerogen-responsiveness among T cells of thymus and spleens harvested periodically from B10.S mice after neonatal inoculation of (B10.AxB10.S) F1 cells. We found that V beta 11+ cells were selectively eliminated within 1-2 days from the thymus glands of all recipient mice, B10.A-specific proliferative T cells disappeared concordantly, and the thymus glands contained donor-derived chimeric cells. Subsequently, an increasing proportion of injected mice acquired V beta 11+ thymocytes and B10.A-responsive T cells, while losing chimeric cells. However, deletion and in vitro nonreactivity were still apparent in the majority of mice at 60 days of age. We conclude that neonatal inoculation of I-E-expressing cells established thymic chimerism and caused profound elimination of V beta 11+ cells from the thymus glands of all injected mice. Therefore, the failure of the majority of neonatally injected B10.S mice to display transplantation tolerance of B10.A antigens as adults cannot be ascribed to an inability to delete I-E-reactive thymocytes.
Mice of the B10 background that are class II I-E nonexpressing demonstrate a relative resistance to neonatal induction of tolerance of class I alloantigens from I-E-expressing B10 strains. The majority of these injected mice delete donor-responsive and "I-E-reactive" (V beta 11+) cells in the immediate postinoculation period, with many remaining deleted of donor-specific T cells before the application of the test skin graft. Utilizing a hemisplenectomy technique in B10.S (I-E-) mice that received as neonates MHC-disparate (B10.SxB10.A)F1 (I-E+) lymphohematopoietic cells, we determined the proportion of adult mice that demonstrated pretransplant donor cell chimerism as well as several functional and phenotypic features ascribed to donor responsiveness. Surprisingly, chimeric cells were present in the bulk of recipients and many also exhibited a deletion of V beta 11+ T cells and a lack of alloreactivity to the donor strain allotype. Since chimeric, MLR-, V beta 11-deleted mice would be predicted to be tolerant to allograft challenge, we hypothesized that the test skin graft applied in adulthood was providing stimulatory signals that overcame this state of immunologic unresponsiveness. To examine this issue, injected mice that had been evaluated before skin grafting were challenged with donor-specific skin and evaluated for the same parameters measured in the pretransplant period. The majority (95%) of these mice subsequently rejected the B10.A skin graft in a range of 7-14 days. After graft rejection, V beta 11+ cell levels generally increased and chimeric cells were typically eliminated. Thus, the ability to reject allografts is not predicted by a "nonresponsive" immune phenotype or the presence of chimeric cells before application of the test allograft. In fact, the graft appears to provide an in vivo stimulus to the reduced numbers of host donor-specific T cells that results in the removal of chimeric cells and a breakdown of the tolerant state. We conclude that application of orthotopic skin grafts provides the signal(s) necessary to break class I tolerance induced neonatally in the context of I-E disparity.
Mycobacterium tuberculosis grew, from a bronchoalveolar lavage of a patient with AIDS, on a gentamicin-supplemented cell culture monolayer, causing a focal and slowly spreading cytopathic effect resembling that of a virological isolate. The same effect was observed after inoculation of two different inocula of M. tuberculosis onto the same cell culture.
Uninsured and underinsured persons in the United States have limited access to health and mental health care and often enter the health care system via hospital emergency rooms. This pattern is even more pronounced among ethnic minorities, particularly Hispanics, a disproportionate number of whom are uninsured. The author reviews the problems faced by Hispanics in gaining access to the health and mental health care system and examines the current health and mental health status of the Hispanic population. He suggests public policy initiatives to increase the likelihood that currently uninsured members of ethnic minorities, including Hispanics, will have access to high-quality health and mental health care.
Some reports have stated that central norepinephrine (NE) depletion inhibited the development of hypertension in the rat. On the other hand, this pharmacological treatment induces changes on the central renin-angiotensin system. The present study was designed to follow the development of 2 kidney-2 clip (2k-2c) renovascular hypertension in rats depleted of central NE and to analyze the central and peripheral renin-angiotensin system. Male Wistar rats (n = 40) were used. Half of the animals was injected, intracisternally, with 6-hydroxydopamine (6-OHDA), the remaining rats only received the vehicle. One week later a silver clip was placed on each renal artery on half of the 6-OHDA treated rats and on half of the vehicle treated animals. A sham operation was performed on the remaining rats. Blood pressure was measured weekly during 7 weeks. Then, blood and cerebrospinal fluid (CSF) samples were obtained. The brain was dissected in several areas. NE and angiotensinogen concentration (AoC) were determined in tissue samples. AoC was evaluated in plasma and CSF; plasma renin activity was also measured. Hypertension development was not prevented by central NE depletion, which was significant in all central areas (p < 0.001). Other significant results showed that renal ischemia and/or NE depletion induced a significant increase in angiotensinogen concentration in the hypothalamus (p < 0.01) and in CSF (p < 0.05). In summary: central NE depletion was not able to modify the development of 2 k - 2 c hypertension. Treatment and renal ischemia induced an increase of central AoC.
The rat monoclonal antibody (mAb) termed EA-1 was originally selected for its capacity to block the adhesion of T lymphocyte progenitors to mouse thymic endothelium. Here we show that the mAb EA-1 recognizes the alpha 6 chain of alpha 6 beta 1 and alpha 6 beta 4 integrins. Both molecules are present at a high level on the luminal and basolateral side of vascular endothelium and alpha 6 beta 1 integrin is expressed on the highly metastatic cell lines B16/129 (melanoma) and KLN-205 (carcinoma). These lung specific tumors bind preferentially to lung frozen sections, and EA-1 blocked this interaction in vitro. Moreover, mAb EA-1 inhibited experimental metastasis to the lung of B16/129 cells injected intravenously. Metastasis in vivo was blocked when the antibody was injected into mice before or simultaneously with the melanoma cells, as well as when melanoma cells were precoated with EA-1 before injection. We suggest that alpha 6 integrins play a dual role in the metastatic process, mediating the adhesion of tumor cells to the luminal surface of the endothelium and the adhesion to laminin in the subendothelial extracellular matrix during extravasation. Despite the fact that alpha 6 integrins are laminin receptors, EA-1 did not interfere with melanoma cell binding to laminin fragments. Our antibody EA-1 may therefore recognize a binding domain on alpha 6 integrins of a novel ligand involved in cell-cell interaction.
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The present experiments were designed to assess a possible role of H2O2 in the proliferation of cultured rat mesangial cells, as well as to evaluate the effect of different calcium channel blockers and a platelet-activating factor antagonist on this proliferation. Cultured rat mesangial cells were plated at two densities (10,000 and 25,000 cells/well) in 24-well dishes, and proliferation was measured by analyzing [3H]thymidine incorporation and by directly counting the cells. Hydrogen peroxide, 100 microM, increased [3H]thymidine incorporation at the two densities tested (85 and 59%, respectively), as well as the number of cells (53 and 23%, respectively). This effect was dose dependent and it was blocked completely by verapamil and diltiazem, 10 microM, but not by TMB-8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester] at the same concentration. BN 52021, a competitive antagonist of platelet-activating factor, only slightly blocked the H2O2-dependent proliferation. The inhibitory action of the two calcium antagonists tested started at concentrations as low as 1 nM, and inhibited completely the H2O2 stimulated proliferation at concentrations between 0.1 and 1 microM. These results establish that H2O2 is able to induce proliferation of mesangial cells. Although the pathophysiological implications of this finding remain to be proven, these data suggest a potential therapeutic action of calcium antagonist in inflammatory conditions such as glomerulonephritis.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor being used increasingly to support white blood cell counts in hematologic disorders. Since the survival of IgG-sensitized cells following blood transfusions and the clearance of immune complexes are important in these disorders, we investigated the effect of GM-CSF on the Fc gamma receptors largely responsible for this immune clearance. Human monocytes were cultured in buffer or 100 U/mL of recombinant GM-CSF (rGM-CSF) for 48 hours. Flow cytometry was used to evaluate changes in the expression of the three Fc gamma receptors. Fc gamma RII was the only Fc gamma receptor significantly increased by rGM-CSF. This increase in Fc gamma RII surface protein was correlated with an increase in macrophage binding of erythrocytes sensitized with IgG. In addition, an increase in monocyte binding of IgG-sensitized RBCs was observed in RBCs sensitized with murine IgG2b antibody, which preferentially binds to Fc gamma RII. rGM-CSF also increased the monocyte Fc gamma RII-dependent low-affinity binding site for trimeric IgG. Furthermore, rGM-CSF was observed to increase the expression of monocyte Fc gamma RII mRNA, including that for Fc gamma RIIA. Thus, these studies demonstrate that GM-CSF increases monocyte Fc gamma RII expression and function and suggests that a similar process may be present in vivo. This effect may be either beneficial (increased clearance of immune complexes) and/or detrimental (increased transfusion requirements) in select patients.
Renal thromboxane (Tx) production is increased in the MRL-lpr murine model of lupus nephritis. To investigate the relationship between increased Tx production and number and affinity of Tx receptors, we measured binding of the Tx receptor antagonist [3H][SQ295481S-1 alpha,2 beta(5Z),3 beta,4 alpha]-7-(3-((2-((phenyl- amino)-carbonyl)hydrozino)methyl)-7-oxabicyclo-(2.2.1)heptan -2-yl)-5-heptenoic acid in glomerular preparations from MRL-lpr mice and both MRL(-)+/+ and LG/J controls. Renal Tx binding was first characterized in normal LG/J mice. In these animals, glomerular binding was specific, saturable and reversible. Scatchard analysis revealed a single class of high-affinity binding sites. We next evaluated Tx production and binding in 12- and 16-week-old MRL-lpr mice and MRL(-)+/+ controls. To assess renal Tx production, excretion of TxB2 was measured in urine. Urinary TxB2 was increased in MRL-lpr mice at 16 weeks of age. This increase in urinary TxB2 was associated with a reduction in density of glomerular Tx binding sites compared to either 12-week-old MRL-lpr mice or MRL(-)+/+ controls. Ligand binding affinity was similar in all groups. To investigate if this alteration in binding was specific for Tx, glomerular binding of [3H]angiotensin II was measured. In MRL-lpr mice, the number and affinity of glomerular angiotensin binding sites were similar at 12 and 16 weeks of age. Thus, in this murine model of lupus nephritis, enhanced renal Tx production is temporally associated with a decrease in glomerular Tx binding sites without a change in receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
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In MRL mice, the mostly recessive lpr mutation results in both the accumulation of CD4-, CD8-, CD3+ T cells in lymphoid tissue and many features of generalized autoimmune disease, including immune complex glomerulonephritis. To positionally clone the lpr mutation and analyze the effects of background genes, backcross offspring were examined from the cross: (MRL/MpJ-lpr x CAST/Ei)F1 x MRL/MpJ-lpr. The lpr gene was found to be closely linked to a mouse chromosome 19 marker defined by a variation of a Fas gene restriction fragment. Our results identified differences in RNA expression and differences in the genomic organization of the Fas gene between normal and lpr mice, and confirm the recent report that a mutation in the Fas apoptosis gene is the lpr mutation. However, our results also indicate that the Fas gene is expressed in spleen cells from normal mice, and spleen and lymph node cells from mice with a second mutation at the lpr locus (lprcg). Together these results suggest that altered Fas transcription results in the failure of lymphocytes to undergo programmed cell death and may lead to an altered immune cell repertoire. This mechanism may explain certain central and peripheral defects in tolerance that are present in autoimmune disease. The current study also demonstrates the profound effect of background genes on the degree of nephritis, lymphadenopathy, and anti-DNA antibody production. Of major note, our studies suggest the identification of chromosomal positions for genes that modify nephritis. Analysis of the backcross mice for markers covering most of the mouse genome suggests that over 50% of the variance in renal disease is attributable to quantitative trait loci on mouse chromosomes 7 and 12. Moreover, this study provides a model for dissecting the complex genetic interactions that result in manifestations of autoimmune disease.
L-selectin is a cell surface receptor on granulocytes, lymphocytes and monocytes that is responsible for the initial attachment of leukocytes to endothelium. The extracellular domain of L-selectin is proteolytically shed from leukocytes following cellular activation in vitro. The shed form of L-selectin (SL-selectin) is functionally active and at high concentrations can inhibit leukocyte attachment to endothelium. Therefore, an ELISA was developed to quantitate the levels of SL-selectin in biological fluids, biopsy specimens and during recombinant protein production. This simple, quantitative sandwich ELISA uses two monoclonal antibodies directed against the extracellular domain of SL-selectin. The assay has a detection range of 5-1300 ng/ml, is precise and sensitive. The ability of this assay to detect SL-selectin in serum, plasma, and culture supernatant fluid was demonstrated and it was used to quantitate circulating SL-selectin in normal and patient sera. Patients with sepsis and HIV infection showed markedly elevated SL-selectin levels in serum. Thus, the ELISA should prove useful both for laboratory purposes as well as in the diagnostic evaluation of patients with inflammatory diseases.
Cyclic AMP (cAMP) is known to play a key role in regulating insulin action, and it is well documented that in several cases of physiological insulin resistance its concentration is increased. Since late pregnancy in the rat is associated with liver insulin resistance, we have studied possible alterations of some cellular mechanisms regulating the cAMP metabolism. (1) Liver cAMP concentration was shown to be increased by some 30% and 50% at 18 and 22 days of pregnancy respectively, compared with virgins. (2) Basal adenylate cyclase activity was higher only in the 18-days-pregnant rat, and the forskolin-stimulated maximal activity was similar in the three groups of animals. (3) alpha s protein is decreased in term-pregnant rats; however, coupling between Gs and adenylate cyclase is only impaired in the 18-days-pregnant animals, and stimulation by glucagon is impaired in both groups of pregnant animals. (4) Gi-2 protein was shown to be unable to elicit the tonic inhibition of adenylate cyclase in pregnant rats, although it was only decreased at 22 days of gestation. The increased alpha i-2 level detected by immunoblotting at 18 days of gestation did not correlate with its decreased ADP-ribosylation, suggesting that the protein is somehow modified at this stage. (5) Pregnancy is associated with a decrease in membrane phosphodiesterase activity. Our results show that late pregnancy is associated with increases in liver cAMP levels that might be involved in eliciting the characteristic insulin-resistant state, and suggest that mechanisms leading to these increments are changing during this phase of gestation.
Phosphoglycerate mutase consists of two kinds of different subunits, M and B. We previously sequenced a rat cDNA encoding the type-M subunit. Here, we report the sequence of the type-B subunit-encoding cDNA. This cDNA has 1754 bp and contains a long 3'-untranslated region of 897 bp.
Granulocyte Fc gamma receptors are important components in the recognition of IgG-coated cells and immune complexes. Two proteins have been identified on resting human granulocytes which function as Fc gamma receptors, Fc gamma RII (CD32) and Fc gamma RIII (CD16). A third protein, Fc gamma RI (CD64), is not constitutively expressed on resting granulocytes, but can be induced by activation with gamma-interferon. We examined the role of these three Fc gamma receptors on human granulocytes in the binding of both IgG-sensitized erythrocytes and soluble oligomeric IgG. In these studies we employed anti-Fc gamma receptor antibodies which complete for the Fc gamma RII and Fc gamma RIII ligand binding sites. Preincubation of granulocytes with saturating concentrations of high-affinity anti-Fc gamma RII monoclonal antibody did not alter the recognition of IgG sensitized human cells by granulocytes. Furthermore, ligand binding studies demonstrated that anti-Fc gamma RII antibody altered neither the number nor the affinity of granulocyte binding sites for human trimeric IgG. In contrast, Fab anti-Fc gamma RIII inhibited the binding of both IgG (anti-D) sensitized human RBCs and IgG sensitized sheep RBCs. Similarly, a reduction in the expression of Fc gamma RIII by treatment with phosphatidyl-inositol specific phospholipase C reduced PMN recognition of IgG-sensitized cells. Also, anti-Fc gamma RIII decreased the number of granulocyte binding sites for human IgG trimer without a change in receptor affinity. Fc gamma RI, which was induced by gamma-IFN, increased granulocyte recognition of both IgG sensitized RBCs and IgG trimer. These data suggest that Fc gamma RIII is the primary Fc gamma receptor on granulocytes which recognizes IgG sensitized RBCs and low molecular weight complexes of IgG. With gamma-interferon activated granulocytes, Fc gamma RI appears to enhance this recognition process.