A model system of coupled activity of co-immobilized creatine kinase and myosin.

Myosin and creatine kinase were co-immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M-line of myofilaments. The Mg-ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg-ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg-ADP between pyruvate kinase and creatine kinase either free in solution or co-immobilized with myosin was studied at various creatine phosphate concentrations. Bound creatine kinase competed efficiently when present in very low amounts, corresponding to an activity ratio higher than 1:20,000 between creatine kinase and pyruvate kinase and a molar ratio higher than 1:1000 between creatine kinase and myosin. The Mg-ADP produced by myosin ATPase in the vicinity of the film did not diffuse into the bulk solution but, in the presence of creatine phosphate, was recycled into Mg-ATP by the neighbouring creatine kinase. The existence of an unstirred layer near the surface of the film is sufficient to explain the channeling of ADP (or ATP) between co-immobilized myosin and creatine kinase, without direct interaction or 'intimate coupling' between the enzymes. The problem now is to determine the importance of this kind of facilitated diffusion in the myofilaments in vivo.


Differential effects of ketamine enantiomers on NMDA receptor currents in cultured neurons.

The effects of R- and S-ketamine on N-methyl-D-aspartate receptor-activated cation currents (NMDA receptor currents) of voltage-clamped cultured rat hippocampal neurons were investigated using the whole-cell patch-clamp technique. Both enantiomers exhibited a voltage- and use-dependent blockade of NMDA receptor currents, with the S-enantiomer being about twice as potent as the R-enantiomer. Calculated relative forward and backward rates suggest that conformational differences influence the dissociation from the binding site more than the association with it.


Suspected distinct activation pathways of human lymphocytes induced by antilymphocyte globulin and anti-CD3 monoclonal antibody result in different secretion of hematopoietic colony-stimulating activities.

We evaluated the activation sequence of peripheral blood lymphocytes from healthy donors using different mitogens, including antilymphocyte globulin (ALG), anti-CD3 monoclonal antibody (OKT3), and phytohemagglutinin (PHA). Blood mononuclear cells stimulated by ALG, OKT3 and PHA incorporated 3H-thymidine in the same way. When enriched T cells were tested in the presence of interleukin-1 alpha (0 to 100 U/ml, incorporation of 3H-thymidine was greater in those cells stimulated by ALG than by PHA. OKT3 did not activate enriched T cells. Thymidine incorporation was reduced to less than 50% of maximum concentrations by the addition of 10(-7) mol/1,25-dihydroxyvitamin D3 (vit D3) in PHA- or OKT3-activated cells. However, the inhibitory effect of vit D3 was not apparent in ALG-activated cells. Production of granulocyte-macrophage colony-stimulating factor and interleukin-3 by lymphocytes upon activation was consistently higher when cells were treated with ALG or PHA than with OKT3. Taken together, the data indicate that there appear to be distinct functional mechanisms between ALG- and OKT3-induced lymphocyte activation that lead to characteristic immunohematologic events.


Angioscopy: a new light on peripheral vascular disease.

Although fibre-optic imaging has been used in a number of medical specialties for several years, it has only recently begun to develop in the area of peripheral vascular disease. This review considers the historical development of angioscopy, its current role in peripheral vascular surgery and, finally, its potential for the future.


Graft replacement of post-traumatic thoracic aortic aneurysm: results without bypass or shunting.

From 1986 to 1991 13 cases of post-traumatic thoracic aneurysm were treated at our department. All patients had apparent thoracic injury at the time of trauma, and their mean age was 35 years. The mean time between trauma and operation was 3 years and six patients were asymptomatic. In all patients the diagnosis was made by computed tomography and angiography and all post-traumatic thoracic aneurysms were located at the aortic isthmus. No spinal cord protection by bypass or shunting was used during surgery and the clamp-and-repair method with a mean clamping time of 38 min was used in all 13 patients. No renal or neurological complications were observed postoperatively and there were no hospital deaths. The data of 202 patients who had been operated upon for post-traumatic thoracic aneurysms since 1981 have been reviewed with regard to the relationship between spinal cord protection and the incidence of postoperative paraplegia. Different methods of spinal cord protection were used in 121 patients resulting in paraplegia rate of 1.6%. In 81 patients the clamp-and-repair method was used and no case of paraplegia was observed in this group.


The acute thrombogenicity of an infection-resistant rifampicin-soaked Dacron graft: an experimental study in sheep.

Every effort to reduce synthetic graft infection is welcome and when designing antibiotic-bonded grafts it is important not to increase graft thrombogenicity. In order to study the acute thrombogenicity of rifampicin-soaked gelatin-sealed Dacron grafts compared with untreated gelatin-sealed Dacron grafts, an experimental carotid artery sheep model was used. Twenty sheep were anaesthetised and 7-cm-long 5-mm-wide externally supported gelatin-sealed knitted Dacron grafts were sutured end to end into each carotid artery after excising a portion of that vessel. Test grafts had previously been immersed for 15 min in a rifampicin solution (1 mg ml-1) while control grafts were immersed in physiological saline for 15 min. There were two groups with 10 sheep in each. In one group the blood flow through the grafts was unrestricted but in the second the flow was restricted to 25 ml min-1. Platelets from sheep labelled with 111In and sheep fibrinogen labelled with 125I were injected intravenously. The isotope activities were continuously measured proximally and distally over the grafts for 4 h. With unrestricted flow 4 out of 10 rifampicin-soaked grafts occluded compared with 2 out of 10 control grafts (N.S.). Time to occlusion, thrombus weight, platelet and fibrinogen activity did not differ. In the restricted flow group 9 out of 10 rifampicin-soaked grafts occluded compared with 6 out of 10 control grafts (N.S.). The time to occlusion did not differ. The thrombus weight in the rifampicin group was significantly higher compared with the control group.(ABSTRACT TRUNCATED AT 250 WORDS)


Gastroschisis: what part can the obstetrician play?

In order to assess the contribution of antenatal diagnosis to the management of gastroschisis, the antenatal and postnatal data from 24 cases of gastroschisis observed between 1974 and 1990 were re-examined. The frequency of antenatal diagnosis (29%), polyhydramnios (21%), atresia of the small intestine (29%), length of pregnancy (36.6 +/- 2 weeks of amenorrhea), proportion of cesarean sections (21%), birth weight (2230 +/- 430 g) and the Apgar score after 5 min (9.0 +/- 1.5) were comparable to findings reported elsewhere. The overall mortality rate for the period studied was high (41%), and was not influenced by any antenatal parameter or by the mode of delivery; its recent fall (P = 0.01) is explained by the development of therapeutic methods. These results are consistent with other recent studies and suggest that antenatal diagnosis does not enable an accurate assessment of the prognosis to be made and has no major influence on the mortality rate in gastroschisis.


[Age-related changes in glucocorticoid and mineralocorticoid receptors in lymphocytes of healthy persons and patients with hypertension].

It has been found that the number of glucocorticoid receptors in lymphocytes of the peripheral blood of healthy elderly subjects increases, while the number of mineralocorticoid receptors decreases. The mechanisms of hormone-receptor interactions in hypertension are activated: the number of glucocorticoid and mineralocorticoid binding sites grows in hypertensive patients. Still a more essential rise in the number of receptors is observed in mid-age hypertensive patients than in elderly ones.


The response of uterine fibroids to GnRH-agonist treatment can be predicted in most cases after one month.

Twenty-seven patients with uterine fibroids were treated for 3 months with the GnRH-agonist goserelin prior to surgical myomectomy. Ovarian function was suppressed reliably in all patients. After three applications, 15 fibroids were reduced in volume by more than 50%, and one complete remission was achieved. Seven patients showed a decrease of 10-50% in volume. However, in 5 cases there was no significant reduction. Analysing the time course of the fibroid reduction, the response can be predicted in most cases as early as four weeks after the first injection. Retrospective statistical analysis showed that a 50% reduction in fibroid size due to GnRH treatment is preceded by a 35% reduction after 4 weeks in 81% of cases, and after 8 weeks in all cases. Only 2 of 12 fibroids, which showed a smaller response (less than 50%) to GnRH therapy, were reduced by more than 35% after 4 and 8 weeks. In most cases it seems to be possible to estimate the individual response to GnRH-application after the first injection, so that it is possible to stop therapy in non-responding patients.


Altered regulation of renin secretion by insulinlike growth factors and angiotensin II in diabetic rats.

The etiology of the low renin state in DM is not clear. To assess the role of certain growth and regulatory factors in this process, we studied the effects of insulin, IGF-I, and IGF-II on the renin-angiotensin system in normal and 8-wk STZ-induced diabetic rats. Renin secretion was studied both in static incubations and by perifusion of rat renal cortical slices. In diabetic rats, both plasma renin activity (0.65 +/- 1.6 vs. 4.0 +/- 1.2 ng ANG I.ml-1.h-1) and tissue renin concentrations (27 +/- 5 vs. 51 +/- 8 ng ANG I.mg tissue-1.h-1) were reduced. Insulin (0.1-1.0 mu/ml) and IGF-I (10(-9) to 4 x 10(-9) M) stimulated renin secretion in normal tissue (control, 95 +/- 3%; insulin [0.5 mu/ml], 134 +/- 7%; IGF-I [4 x 10(-9) M], 149 +/- 7%). IGF-I stimulated renin secretion in perifusions as early as 30 min, whereas IGF-II had no effect. However, in diabetic renal tissue, neither insulin (0.1-1.0 mu/ml) nor IGF-I (10(-9) to 4 x 10(-9) M) had an effect on renin. This lack of effect was overcome by adding up to 100-fold higher concentrations of these growth factors. ANG II (10(-10) M-10(-8) M) had an exaggerated inhibitory effect on renin secretion in diabetic tissue. This study suggests that the low renin state in DM may be explained by the enhanced inhibitory effect of ANG II and the resistance to the secretogogue actions of insulin and IGF-I.


Tissue-specific regulation of insulin receptor mRNA levels in rats with STZ-induced diabetes mellitus.

In rats with STZ-induced diabetes mellitus, a reduction in insulin secretion is associated with increased insulin binding in the liver, muscle, fat, and kidney, but not in the brain. To test the hypothesis that tissue-specific modulation of insulin receptors (IRs) in STZ-induced diabetes occurs at the level of mRNA, IR mRNA levels were measured in the liver, kidney, and brain of Sprague-Dawley rats 15 days after intravenous administration of STZ (60 mg/kg body weight) and compared with those of control rats. Diabetic rats were either left untreated or given differing insulin regimens that were designed to achieve varying degrees of metabolic control. IR mRNA levels were measured by slot blot hybridization with a 32P-labeled rIR probe and standardized by 28S ribosomal RNA determination. Hepatic IR mRNA levels were increased significantly in both untreated diabetic rats and in those that received low-dose (2 U/day) insulin therapy. In contrast, hepatic IR mRNA levels did not differ significantly from controls in those that received moderate doses of insulin (3-8 U/day) and were significantly less than controls in those that received the highest doses (6-10 U/day). Renal IR mRNA levels also were increased significantly in the untreated diabetic rats but not in those that received low- or moderate-dose insulin therapy, and were significantly less than controls in those that received the highest doses.(ABSTRACT TRUNCATED AT 250 WORDS)


Brain D1 dopamine receptor in alloxan-induced diabetes.

Specific binding of [3H]SCH 23390 to dopamine D1 receptors in the striatum and olfactory tubercle in 14-day alloxan-induced diabetic rats was investigated. The Scatchard analysis revealed decreased D1 receptor density in the striatum (Bmax values were 548 +/- 23 fmol/mg protein for the control and 466 +/- 33 fmol/mg for the diabetic rats). No change was observed in the olfactory tubercle (Bmax; 299 +/- 27 fmol/mg for the control and 317 +/- 32 fmol/mg for the diabetic rats). Thus, specific binding of [3H]SCH 23390 to striatal and olfactory tubercle membranes showed region-specific changes of brain dopamine D1 receptors in alloxan diabetic rats.


Cerebral taurine transport is increased during streptozocin-induced diabetes in rats.

Taurine is a cerebral osmolyte whose intracellular content changes in parallel with plasma osmolality. We conducted experiments to assess whether cerebral taurine transport is modified during chronic hyperglycemia. Rats with STZ-induced diabetes were studied after 1 wk of sustained hyperglycemia. Cerebral taurine uptake in synaptosomes (metabolically active nerve terminal vesicles) was measured using a rapid filtration technique. The synaptosomes were isolated by homogenization of the brain and purification on discontinuous Ficoll gradients (n = 8 synaptosome preparations). Diabetic rats (n = 13) displayed a 15-25% increase in synaptosomal taurine uptake compared with normoglycemic control animals (n = 12) at all time points assayed between 5 and 120 min. Thus, after a 30-min incubation, cerebral taurine uptake increased from a control level of 3.53 +/- 0.23 to 4.10 +/- 0.24 mumol/mg protein (n = 10) in hyperglycemic rats, P less than 0.03. The magnitude of the plasma-to-brain cell taurine gradient was unchanged in diabetic animals. The intrasynaptosomal taurine concentration (approximately 2 microM) and taurine efflux from the synaptosomes were no different in hyperglycemic versus control rats; efflux amounted to less than 2.5% of the uptake value at corresponding time points. Maximal brain taurine uptake under both control and experimental hyperglycemic conditions required the presence of external Na+ and Cl-. Synaptosomal taurine transport was reduced by competing beta-amino acids such as beta-alanine, beta-aminoisobutyric acid, and hypotaurine (P less than 0.01). Addition of oubain and the anionic binding site inhibitors, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'diisothio-cyanatostilbene-2,2'-disulfonic acid, also decreased cerebral taurine uptake under normoglycemic and hyperglycemic conditions (P less than 0.01). The increased synaptosomal taurine uptake by diabetic rats was not a result of generalized membrane dysfunction because glycine transport was not elevated in hyperglycemic rats. The enhanced transport rate was attributable to a 35 and 81% increase in the Vmax of the high- and low-affinity taurine transporters, respectively (P less than 0.01), without significant change in the Km of the carrier systems. Treatment of hyperglycemic rats (n = 5) with ultra-long-acting insulin to normalize the serum glucose concentration restored synaptosomal taurine uptake to the level observed in normoglycemic controls. The effect of insulin was attributable to correction of hyperglycemia, because addition of insulin (500 mU/ml) to the in vitro assay system did not alter synaptosomal taurine uptake.(ABSTRACT TRUNCATED AT 400 WORDS)


Thromboxane-receptor blockade increases water diuresis in cirrhotic patients with ascites.

This study was undertaken to investigate the role of increased renal thromboxane (TX) A2 production in modulating renal hemodynamics and sodium and water retention in cirrhotic patients with ascites. In a randomized, double-blind, placebo-controlled, crossover trial, 15 nonazotemic cirrhotic patients with ascites and elevated urinary TXB2 excretion received the thromboxane-receptor antagonist ONO-3708 (3 micrograms.kg-1.min-1) in a 4-hour continuous infusion. Administration of ONO-3708 significantly blocked TXA2 receptors; bleeding time showed a twofold increase (432 +/- 65 vs. 131 +/- 17 seconds; P less than 0.005), and platelet aggregation to U-46619 (an agonist of TXA2 receptors) was abolished in all patients studied. The drug induced a significant increase in free water clearance (3.06 +/- 0.70 vs. 1.72 +/- 0.57 mL/min; P less than 0.001) and diuresis (4.74 +/- 0.79 vs. 3.94 +/- 0.66 mL/min; P less than 0.05) compared with placebo, as well as a significant (14%) increase in renal plasma flow. The increases in both free water clearance and diuresis induced by ONO-3708 were directly related to basal urinary TXB2 excretion. These results suggest a role for renal TXA2 as a modulator of water handling in cirrhotic patients with ascites.


Effect of omega-3 fatty acids on rectal mucosal cell proliferation in subjects at risk for colon cancer.

The effects of 12 weeks of omega-3 fatty acid supplementation on rectal mucosal proliferation were assessed with [3H]thymidine autoradiography in a double-blind, placebo-controlled study of 20 patients with sporadic adenomatous colorectal polyps. In the group of 10 that received fish oil containing eicosapentaenoic acid (4.1 g/day) and docosahexaenoic acid (3.6 g/day), the mean percentage of replicative "S"-phase cells in the upper part of colonic crypts (considered a reliable marker of colon cancer risk) significantly dropped from the baseline level after only 2 weeks of treatment and remained lower throughout the study period; no change in upper-crypt labeling was observed in the 10 placebo patients. Rectal mucosal eicosapentaenoic acid content increased in fish oil patients, whereas arachidonic acid levels decreased. The fish oil-induced kinetic changes represent contraction of the proliferative compartment to the levels of a low-risk population and may be related to omega-3 fatty acid effects on the arachidonic prostaglandin pathway. In this short-term trial, fish oil appeared to exert a rapid effect that may protect high-risk subjects from colon cancer.


Ontogeny of leukocyte populations in the ileal Peyer's patch of sheep.

Enzyme- and immunohistochemical methods were used to characterize the leukocyte populations present in the ileal Peyer's patches of sheep foetuses between 68 and 135 d of gestation and particularly in the period around 100 d of gestation, when active lymphopoiesis begins. A wide variety of leukocytes including IgM+, CD5+, CD4+, CD8+ cells, and MgATPase+ dendritic cells were present at an early stage. Groups of IgM+ cells were seen immediately beneath the epithelium as early as 70 d of gestation. Conventional morphometric and computer-assisted morphometric techniques were used to confirm the significant expansion of these cell populations from 90 d of gestation. IgM+ and CD5+ cells were responsible for the vast majority of the increase in cell numbers. It was concluded that a diverse leukocyte population was present at the initiation of active lymphopoiesis in the ileal PP of the sheep foetus and that all members of this population were associated with the emergence of the dome/follicle primordia from which the B-cell follicle develops.


Severely disabled elderly persons with financially catastrophic health care expenses: sources and determinants.

This article describes the sources of financially catastrophic health care expenses among disabled elderly persons. Using a cost-to-income approach and data from the 1981-1982 Channeling Demonstration project, we examined the types of health care costs (hospital, physician and ancillary care, nursing home, and prescription medicine) that contributed to overall expenses. For the Channeling sample, out-of-pocket expenses for prescription medicines and for nursing home care were the principle source of catastrophic expenses.


A 28-day feeding study with methyl isoeugenol in rats.

Methyl isoeugenol was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 30, 100 and 300 mg/kg body weight/day. A further group of 16 male and 16 female rats was given the rodent diet as a control. The administration of methyl isoeugenol in the diet did not adversely affect the growth or general health of the animals or their food intakes. Although high dose animals of both sexes had increased lymphocyte and total white blood cell counts, these are not considered, in isolation, to be an adverse effect of treatment. None of the minor variations observed in the serum chemical analyses or urine analyses is considered to be indicative of a treatment-related toxic effect. An increase in liver weight, adjusted for body weight, was seen in male and female rats receiving 300 mg methyl isoeugenol/kg body weight. Few histopathological abnormalities were observed. Although the incidence of kidney and Harderian gland lesions was higher for high dose animals compared with the controls, the lesions are of a type that occurs spontaneously and are thus not considered to be attributable to treatment with methyl isoeugenol. While the increased liver weight and white blood cell counts of rats given 300 mg methyl isoeugenol/kg body weight may represent effects of treatment, it is not considered that there is any reason to regard these as adverse effects.


[Fc gamma receptors: structure, function, and clinical significance].

Fc gamma receptors are a group of three different receptors with several subtypes. They are widely distributed on many cells of the immune system and contribute to the pathogenesis of immune complex- and autoantibody-mediated diseases such as vasculitis, rheumatoid arthritis, idiopathic thrombocytopenic purpura or autoimmune neutropenia. This review focuses on the structure, distribution and function in Fc gamma receptors and their subtypes.


[The GPI-anchored Fc gamma receptor III in the activation of granulocytes].

PMN express primary two Fc gamma receptors for IgG immune complexes. Fc gamma RII is a transmembraneous molecule in contrast to Fc gamma RIII on PMN which is GPI-linked. In spite of the different membrane linkage both Fc gamma R use calcium as an intracellular signal. Cross-linking of Fc gamma RIII is more effective for induction of H2O2 production than Fc gamma RII. Monoclonal IgG1x cryoglobulin complexes activate PMN via Fc gamma RIII as shown by calcium mobilization and H2O2 production.


Specific down-regulation of allograft reactivity at the cellular level: graft cells and responder T cells.

Local immune reactivity in an allograft is largely determined by (1) the immunogenicity of the grafted cells and (2) the presence of recipient allospecific T lymphocytes and, much more importantly, their capacity to react and initiate damage to the graft. Ad (1): It is still widely believed that allograft immunogenicity is sufficiently described by the cell surface expression of transplantation antigens, in particular MHC class I and II molecules. In extension of earlier, more complex models it will be demonstrated that transfected subclones of a macrophage cell line show unaltered, strong MHC expression but have become non-immunogenic and, most importantly, have acquired the capacity to render mature naive T cells anergic in a specific fashion. Ad (2): Starting from the above and from other observations, we have found in human organ graft recipients that cytotoxic T lymphocyte (CTL) frequencies may be specifically down-regulated but may regain their reactivity after cultivation under ex vivo conditions. In keeping with the latter we can now show quantitatively the variable presence and the functional potential of cell-released (soluble) human MHC (HLA class I) molecules: they are able to specifically inhibit human CTL reactivity in a dose-dependent fashion. Taken together, it may be concluded that studies at the cellular level, in particular the level of human cells, will increasingly help us to better understand and manipulate the immunological interplay between the local graft cell and its most important antagonist, the T lymphocyte.


Identification of a 64-kDa protein phosphorylated with glucose in human polymorphonuclear leukocytes in a cell-free system.

We previously reported that a 64-kDa protein (p64) in human polymorphonuclear leukocytes (PMN) was phosphorylated with [gamma-32P]ATP under a micromolar concentration of glucose in a cell-free system. The present paper presents the results of analysis of phosphorylation reaction and the identification of phosphoprotein. The findings that p64 was also phosphorylated with glucose-6-[32P]phosphate and that phosphorylation was inhibited with mannoheptulose suggested that the reaction was mediated by hexokinase. In fact, it was found that [32P]phosphate in glucose-6-[32P]phosphate was incorporated into either p64 or rabbit muscle phosphoglucomutase and that glucose-6-phosphate formation from glucose and ATP was detected in over 100-kDa fraction of PMN cytosol. These results showed that p64 was phosphoglucomutase in PMN and that phosphate incorporation into p64 was a conversion of a phosphate group in glucose-6-phosphate produced by hexokinase. It was further demonstrated by analysis of two-dimensional electrophoresis that p64 phosphorylated with glucose induction was different from another 64-kDa protein phosphorylated by stimulation with formyl-methionyl-leucyl-phenylalanine in vivo.


The amplifier role of T cells in the human in vitro B cell response to type 4 pneumococcal polysaccharide.

The human B cell response to T cell independent type 2 antigens is regulated by thymus-derived lymphocytes. We analyzed the role of T cells in the in vitro antibody response to type 4 pneumococcal polysaccharide (PS4). We here show that the amplifying effect of T cells, which has previously been shown to be radioresistant and confined to T cell preparations enriched for CD4+ cells, is MHC non-restricted as demonstrated in cultures carried out in the presence of allogeneic T cells. Also, T cell clones derived from non-related donors are able to enhance the B cell response to PS4. All TCR alpha beta +, CD 4+ T cell clones, but none of the TCR alpha beta +, CD 8+ T cell clones tested, enhanced the B cell response to PS4. Furthermore, 3 out of 6 TCR gamma delta+ T cell clones were capable of enhancing the anti-PS4 B cell response. Experiments using recombinant lymphokines and glutaraldehyde-fixed T cells indicated that both lymphokines and T-B cell interactions are required for an optimal antibody response to PS4.


Increased phospholipase A2 activity in peritoneal leukocytes in rat experimental colitis.

Activated leukocytes in the peritoneal cavity have been shown to release phospholipase A2(PLA2) activity and increased production of eicosanoids. It is not known whether a similar phenomenon occurs in inflammatory bowel diseases. We measured PLA2 activity in the peritoneal leukocytes and eicosanoid content in the peritoneal cavity of rats with experimental colitis induced by dinitrochlorobenzene, immune complex-mediated colitis, and acetic acid-induced colitis. In all models of colitis, proteins in the peritoneal cavity were increased by 60-260% above control, leukocyte number was elevated by 3- to 10-fold, the PLA2 activity by 4- to 32-fold, and anti-PLA2 activity was absent. Eicosanoid concentrations were 3- to 4-fold higher than controls. In vitro studies confirmed that the peritoneal leukocytes were the source of PLA2. Treatment of rats with dexamethasone and leukocytes in vitro reduced PLA2 significantly, but they were well above control rats without colitis. However, dexamethasone did not improve the histology. These studies suggest that peritoneal leukocytes may serve as an additional source of inflammatory mediators, which may further enhance the inflammatory response in the rat colon.


The Th1/Th2-like switch in syphilitic infection: is it detrimental?

Organisms that cause chronic diseases have evolved mechanisms to evade those immune defenses that resolve the acute stage of infection (10, 12-14, 21, 22, 32, 35, 37, 38, 40, 42, 45-49, 53). Much is to be learned by specifically identifying the mechanisms underlying these evasive strategies. Important new insights will emerge in terms of immunoregulatory pathways. This in turn will facilitate vaccine development. A good example is leishmania infection. The acute stage of this disease is resolved by DTH-macrophage activation. Leishmanial components preferentially activate Th2 lymphocytes. As a consequence, Th1 effects are minimized and infection is exacerbated leading to chronicity (10, 14, 32). To overcome this negative tendency, leishmanial vaccines are administered in combination with exogenous gamma interferon (42). This selects for Th1 predominance and generates protective immunity. Syphilis exhibits many parallels to the other nine chronic diseases mentioned above. Similarities include an acute localized stage that readily heals, early clearance via DTH-macrophage activation, transient concomitant immunity during acute infection, development of macrophage suppression through PGE2 down-regulation, beneficial effects of exogenous gamma interferon, and elements of autoimmunity. Some of the complexities of immunoregulation during treponemal infection have just begun to be unraveled. It will be important to develop further insight into the Th1/Th2 switch especially as it relates to chronicity. Macrophages seem to be intimately involved in the mechanics of this switch, and their specific role needs further clarification. Whatever is learned about syphilis, as well as other chronic infections will contribute to a better understanding of the generalized pathways of immunoregulation.


Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to epithelial cells.


Evidence for actinlike proteins in an M protein-negative strain of Streptococcus pyogenes.

Antigens shared between Streptococcus pyogenes and heart tissue may play an important role in autoimmune cardiac injury associated with acute rheumatic fever. Antiheart/antistreptococcal antibodies found in the disease react with antigens of S. pyogenes, including M protein and a 60-kDa antigen distinct from M protein. Heart antigens recognized by these cross-reactive antistreptococcal antibodies include myosin and actin. To investigate the presence of a streptococcal actin, established protocols for the polymerization and isolation of eukaryotic actin were used to extract and concentrate actinlike proteins from M- streptococcal cells. The polymerized bacterial actin from the streptococcal extract was probed in immunoblots with an antiactin monoclonal antibody. Two proteins of about 60 kDa in the polymerized bacterial actin reacted with the antiactin antibody. Proteins in the polymerized bacterial actin extract of about 43 and 60 kDa behaved like eukaryotic actin by binding to myosin and DNase I affinity columns. Filaments were demonstrated by electron microscopy in the polymerized bacterial actinlike extract, which also enhanced the ATPase activity of eukaryotic myosin. The data suggest that proteins resembling actin are present in S. pyogenes.


Effects of light exposure on the uptake of photofrin II in tumors and normal tissues.

DBA mice bearing CaD2 mammary carcinomas were used to determine the effect of giving small doses of light to the tumor area 1.5 hr after injecting Photofrin II (PII). The smallest light dose applied (12.5 J/cm2) had no effect on the uptake of PII in the tumor and its surrounding tissues, as measured 24 hr after the i.p. injection. However, several higher light doses increased the uptake of PII in the tumor significantly, the uptake in skin slightly, while the uptake in muscle tissue was decreased rather than increased. Thus, the PII concentration ratio between the tumor and the surrounding normal tissues was significantly improved. The rates of clearance of PII from irradiated tissues and non-irradiated tissues were not significantly different. Most likely, the present observations are due to transient pH lowering in the tumor resulting from vascular damage.


Menogaril in the treatment of malignant mesothelioma: a phase II study.

Menogaril is a new semisynthetic anthracycline agent derived from the antitumor antibiotic Nogalomycin. Compared to doxorubicin it has similar or improved activity in anti-tumor cell line screening; human tumor cloning assays suggest modest anti-tumor activity as well. Menogaril is much less cardiotoxic than doxorubicin. We performed a phase II trial of this agent in 22 patients with advanced malignant mesothelioma. At a dose of 200 mg/m2 iv every 4 weeks (160 mg/m2 in previously radiated patients) only 1 of 22 (5%) evaluable patients had a partial remission lasting 4 months. (95% confidence limits 0.1-23%). The major toxic effects included pain at the site of infusion and granulocytopenia. While well tolerated, Menogaril has minimal activity in malignant mesothelioma. We do not plan further studies with Menogaril in this disease.


Toremifene and its metabolites enhance doxorubicin accumulation in estrogen receptor negative multidrug resistant human breast cancer cells.

The enhanced accumulation of doxorubicin by agents known to reverse multidrug resistance provides a good functional test for evaluating modulating activity. In the present study, the non-steroidal triphenylethylene toremifene selectively increased doxorubicin accumulation in multidrug resistant estrogen receptor negative MDA A-1 human breast cells compared to the MDA 231 wild type cells. MDA A-1 cells were noted to be 1,000 fold resistant to doxorubicin (IC 50 = less than 0.1 microgram/ml MDA 231; IC 50 = 100 micrograms/ml MDA A-1). Total accumulation of doxorubicin, expressed as area under the time concentration curve (AUC), was increased significantly in doxorubicin resistant cells (156% increase) versus wild type MDA 231 cells (6% increase). Correction of the accumulation defect to doxorubicin in drug resistant cells required a 18-20 hour pre-incubation with toremifene. The effects of toremifene on cell cycle in MDA A-1 cells was analyzed by flow cytometric techniques. Toremifene had a dose response relationship in blocking cells in G0-G1 reducing the number of cells entering S phase of the cell cycle. This effect was maximal at concentrations which increased the accumulation of doxorubicin in MDA A-1 cells. Several metabolites of toremifene were also noted to increase doxorubicin accumulation in MDA A-1 doxorubicin resistant cells. Tore XVIII (deaminocarboxytoremifene), Tore IV (4-hydroxy-N-desmethyltoremifene) and N-desmethyltoremifene all increased the accumulation of doxorubicin significantly (114%, 128% and 42% respectively). Finally, we show evidence that toremifene and its active metabolites are present in high concentrations in human plasma following a single 200 mg oral dose.(ABSTRACT TRUNCATED AT 250 WORDS)


Early experiences of laparoscopic cholecystectomy in five Irish hospitals. Irish Laparoscopic Group.

There are many concerns about the widespread introduction of laparoscopic cholecystectomy. The initial experience of five hospitals in introducing laparoscopic cholecystectomy was reviewed. Three hundred and eight patients were operated upon, and the operations were completed laparoscopically in 279 (91 percent). One patient sustained a diathermy injury to the right hepatic duct. There was no mortality and the overall morbidity was 10 percent. Mean postoperative stay was 3.6 days. The participating surgeons considered training workshops to be desirable and felt that trainees should be supervised for at least ten cases. Laparoscopic cholecystectomy can be safely introduced and performed, and it should be considered in all patients undergoing cholecystectomy.


Severe hypercalcaemia four months after acute oliguric renal failure--successful treatment with intravenous clodronate.

A 33 year old man developed acute oliguric failure lasting 66 days, eight days after admission with multiple gun shot wounds. On day 99 after admission, serum calcium was elevated mildly at 2.54 mmol/l (normal range 2.1-2.5 mmol/l). Serum parathormone was undetectable. He was discharged soon afterwards. He presented again on day 164 with nausea, vomiting and blurred vision. Fundoscopy revealed an ischaemic retinopathy and extensive keratopathy. Serum calcium was 3.48 mmol/l and serum creatinine 262 umol/l (normal range 40-110 umol/l). Repeat parathormone was undetectable and there was no evidence of myeloma, sarcoidosis or malignancy. Following treatment with intravenous saline and frusemide, serum calcium fell to a nadir of 3.05 mmol/l. On day 168 an infusion of sodium clodronate 300 mg was given. Twenty-four hours later serum calcium was 2.65 mmol/l and 48 hours later calcium was 2.26 mmol/l. Normocalcaemia was maintained for 17 days and severe hypercalcaemia never recurred. This is the first report in which biphosphonates have been successfully used to treat hypercalcaemia following acute renal failure thus obviating the need for further dialysis.


The management of choledocholithiasis during laparoscopic cholecystectomy by sphincter dilatation--initial experience in ten cases.

The intra-operative management of associated common bile duct calculus during laparoscopic cholecystectomy in ten patients is outlined. The techniques employed to remove these calculi are described. The role of such techniques in the management of common bile duct stone is discussed.


An anatomical study of a new method for enlargement of narrowed aortic annulus: intra-arterial aorto-infundibuloplasty.

A new procedure "intraarterial aorto-infundibuloplasty" for the narrowed aortic annulus is described. Aortic valve replacement is performed through an aorto-pulmonary and infundibular septal incision which is eventually enlarged by a single patch, i.e., a two-dimensional patch instead of a three-dimensional patch as in Konno's procedure. An anatomical study showed that a prosthetic valve three sizes larger than the natural annular diameter could be implanted and the natural annular diameter was increased by as much as 42%.


Serotonergic medications for sexual obsessions, sexual addictions, and paraphilias.

Paraphilias and related disorders have recently been thought of as sexual addictions. However, it has also been argued that these disorders are sexual compulsions. The question arises as to whether these disorders and obsessive compulsive disorder respond in the same way to pharmacotherapy. We retrospectively reviewed outcome in 13 patients who presented with sexual symptoms and were treated with serotonin reuptake blockers. Symptoms were divided into paraphilias, nonparaphilic sexual addictions, and sexual obsessions. Paraphilias had the least improvement, while sexual obsessions had the best response to medication. Paraphilias and related disorders may be less responsive than sexual obsessions or compulsions to serotonin reuptake blockers. Perhaps paraphilias and related disorders are on the impulsive rather than the compulsive end of the spectrum of obsessive compulsive disorders. Controlled trials are, however, necessary to replicate these preliminary findings.


Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes.

A procedure was developed for isolating Chlamydomonas outer-arm dynein that can functionally combine with the axoneme of an outer-arm-missing mutant, oda1. Previous studies showed that the outer-arm dynein of this organism, containing three heavy chains (alpha, beta, gamma), dissociates upon extraction with a high-salt-concentration buffer solution into an 18-S particle containing the alpha and beta heavy chains and a 12-S particle containing the gamma heavy chain. It was found, however, that the three heavy chains did not dissociate if the high-salt extract was centrifuged in the presence of Mg2+; the three chains constituted a single species (23-S dynein) sedimenting at about 23 S and displayed a three-headed bouquet configuration in electron micrographs. Furthermore, the 23-S dynein had the activity to bind to the axonemes of oda1 and increase the reactivated motility of detergent-extracted cell models; its addition increased the beat frequency from 28 Hz to 53 Hz, a frequency comparable to that of wild-type axoneme. The 18-S and 12-S dyneins, on the other hand, were unable to increase the motility of oda1 axonemes even when added together. The new protocol thus enables purification of outer-arm dynein that retains its functional activity. It will provide a useful experimental system with which to study the mechanism of outer-arm function.


Role of 17-kDa essential light chain isoforms of aorta smooth muscle myosin.

Aorta smooth muscle myosin contains two kinds of 17-kDa essential light chain, LC17nm (nonmuscle-type) and LC17gi (gizzard-type) [Hasegawa, Y., Ueda, Y., Watanabe, M., & Morita, F. (1992) J. Biochem. 111, 798-803]. The LC17 isoforms were released from porcine aorta myosin by incubation at 46 degrees C. The rate of release was 1.5 to 2 times higher with LC17gi than with LC17nm. Aorta myosins containing the two LC17 isoforms in various ratios could be reconstituted. The actin-activated ATPase activity was measured as a function of LC17nm content. The Vm value was lower with myosin which contained more LC17nm. The apparent dissociation constant for F-actin, Km, was 20-fold less with myosin which contained 81% LC17nm than myosin which contained 23% LC17nm. A similar difference in the dissociation constants of myosin for F-actin was observed in the presence of adenylyl imidodiphosphate. The role of LC17nm appears to be to make aorta myosin suitable for maintaining the muscle tension with a low expenditure of energy. The isoform-dependent difference in the F-actin-binding affinities of myosin seems partly due to the difference in the affinities of LC17 isoforms themselves for F-actin. We found that the isolated LC17nm itself could bind with F-actin with a dissociation constant of 64 microM, but LC17gi could not.(ABSTRACT TRUNCATED AT 250 WORDS)


Stable and slow-turning-over microtubules characterize the processes of motile epithelial cells treated with scatter factor.

The turnover of microtubules was studied in the processes of PtK2 cells, after treatment with the cytokine scatter factor (SF), using micro-injected biotin-tubulin as a reporter of new microtubule growth. Cells treated with SF became dispersed and fibroblast-like in morphology, showing one or more elongated processes. These processes contained bundles of microtubules, a significant proportion of which did not turn over during incubation times of up to an hour. Short broken pieces of microtubule were frequently found in all parts of the cell, particularly after longer incubation times, suggesting that more-stable microtubules were cut into pieces, which were subsequently degraded. From about half an hour after injection small tangles of stable microtubules were found. Some of these were clearly within the cell bodies. Others were usually larger in size and seemingly located outside the injected cells. These were considered to have formed part of small 'feet' presumed to be broken off during the retraction of trailing processes. The microtubules within the processes were resistant to the effects of both microtubule-depolymerizing drugs and cold under conditions where the processes were maintained. When these microtubules disappeared as the result of longer drug treatment the processes were also lost although, rarely, short processes lacking microtubules were found. It is concluded that the stable microtubules have a major role in process maintenance, although one that is indirect rather than a structural relationship.


Cyclin A-cdc2 kinase does not trigger but delays cyclin degradation in interphase extracts of amphibian eggs.

Purified cyclin B-cdc2 kinase has been shown previously to trigger cyclin degradation in interphase frog extracts by initiating a cascade of reactions that includes cyclin ubiquitinylation and ends with proteolysis. However, cyclin A-cdc2 kinase was not assayed in these early experiments. Here we have shown that full-length recombinant human cyclin A failed to induce cyclin degradation when it was added to frog extracts free of cyclin B, although it formed an active kinase complex with Xenopus cdc2. A highly purified kinase complex containing a truncated human cyclin A and starfish cdc2 also failed to switch on the cyclin degradation pathway. In contrast, both recombinant cyclin B and highly purified cyclin B-cdc2 kinase readily triggered degradation of both cyclins B and A in frog extracts. Whilst free cyclin A had no inhibitory effect, cyclin A-cdc2 kinase delayed degradation of both cyclins A and B induced by cyclin B-cdc2 kinase. The finding that cyclin A-cdc2 kinase cannot turn on, and even delays, cyclin destruction may be essential to prevent premature inactivation of MPF (maturation-promoting factor) before complete condensation of chromosomes and formation of the metaphase spindle.


Immunofluorescence study of actin, acrosin, dynein, tubulin and hyaluronidase and their impact on in-vitro fertilization.

Using polyclonal antibodies, the distribution of actin, acrosin, dynein, tubulin and hyaluronidase has been examined by indirect immunofluorescence in sperm preparations from fertile donors and in-vitro fertilization (IVF) patients. After recording sperm parameters in native semen, spermatozoa were washed free of seminal plasma using either the swim-up or the Percoll filtration technique. Prior to insemination, aliquots of the washed sperm suspensions were prepared for antibody staining. Spermatozoa from fertile donors were analysed in order to establish the specific fluorescence patterns of each antibody and the threshold scores of normality. Immunofluorescence scores obtained from IVF patients were then analysed with respect to IVF outcome. For each tested protein, the number of normal samples were significantly lower in the group which did not fertilize and fertilization rates were significantly reduced when any of the tested proteins were scored as pathological. Normal fluorescence scores were correlated with morphology, motility, velocity and to a lesser extent with sperm concentration in native semen. On the basis of receiver-operating characteristic curves, likelihood ratios and Cohen's kappa values, the presence of acrosin and tubulin yields the most useful information on sperm functional and structural status and on its fertilizing ability.


Distribution of plasminogen and plasmin in fractions of bovine milk.

The relative amounts of immunoreactive plasminogen and active plasmin in different fractions of bovine milk were examined. Raw milk was centrifuged to separate skim, cream, and a somatic cell pellet. Skim milk was centrifuged to separate milk serum and casein micelles. Milk fat globule membranes were isolated from the cream fraction of bovine milk. Proteins from somatic cells were isolated following sonication of the cells. Western blot analysis showed the presence of several forms of plasminogen in bovine milk. The predominant forms of plasminogen identified following electrophoresis under nonreducing conditions were proteins with approximate molecular weights of 88,000, 152,000, and 160,000. The predominant forms of plasminogen identified after electrophoresis under reducing conditions were two proteins with approximate molecular weights of 88,000 and 50,000. The highest amount (82% of the total plasminogen), as determined by an ELISA, was associated with the casein fraction. Lower plasminogen concentrations were associated with the serum, cream fractions, and milk fat globule membranes. The SDS-PAGE of the cream and milk fat globule membranes indicated that some casein was present in both fractions. Thus, the low plasminogen concentrations in these fractions may be associated with the caseins there. No immunoreactive plasminogen was present in the somatic cells. Active plasmin was present in the same milk fractions in which plasminogen was detected: casein, serum, and cream.


Identification of a receptor on the vitelline membrane of bovine oocytes that recognizes bovine immunoglobulin G.

In order to investigate whether Ig molecules affect bovine fertility, bovine follicular oocytes were tested for an oolemmal receptor that recognized IgG. The zona pellucida was removed from 296 oocytes. The zona-free oocytes were then incubated with purified bovine IgG. its Fc fragment, or its Fab fragment. They were subsequently washed and incubated with the appropriate rabbit antibovine IgG (whole IgG-specific, Fc-specific, or Fab-specific), which was covalently attached to polyacrylamide beads (immunobeads). Bead attachment was scored using light microscopy. Bovine IgG and bovine IgG-Fc bound to the oocytes, whereas the IgG-Fab exhibited negligible binding. The data indicate that a bovine oolemmal receptor exists that binds IgG by recognizing the molecule in the region of the Fc-Fab junction. Thus, the binding of IgG or other Ig molecules to bovine oocytes may play a role in sperm and egg interaction.


Sociotropy and autonomy: relationship to antidepressant drug treatment response and endogenous-nonendogenous dichotomy.

This study evaluated the relationship of sociotropic and autonomous personality traits with response to pharmacotherapy for 217 depressed outpatients using the Sociotropy-Autonomy Scale. Sociotropy was related to nonendogenous depression, whereas autonomy was related to endogenous depression. Subjects who had high autonomous-low sociotropic traits showed greater response to antidepressants (and greater drug-placebo differences) than those who had high sociotropic-low autonomous traits (who showed no drug-placebo differences). Hierarchical multiple regression analysis showed that the sociotropy-autonomy, but not the endogenous-nonendogenous, distinction was a predictor of drug treatment response. The combination of endogeneity and autonomy predicted response to placebo. If replicated, these findings may enable better matching of patient traits to various treatment modalities for depression.


Ipratropium bromide aqueous nasal spray for patients with perennial allergic rhinitis: a study of its effect on their symptoms, quality of life, and nasal cytology.

Ipratropium bromide is an anticholinergic agent with topical activity that has been studied as a freon-propelled aerosol spray for therapy of nonallergic rhinitis. This is the first report of its use both as an aqueous nasal spray and in perennial allergic rhinitis. In this study 123 patients who had symptoms of perennial allergic rhinitis were randomized to receive ipratropium bromide 21 micrograms or 42 micrograms or placebo, one spray per nostril three times a day for 4 weeks. Patients maintained daily diaries of duration and severity of nasal symptoms and were evaluated weekly. Mean duration and severity of rhinorrhea was decreased in both ipratropium bromide treatment groups by comparison with placebo, with consistently greatest improvement in the group treated with ipratropium bromide 42 micrograms per nostril three times a day. No statistically significant differences occurred among treatment groups in duration or severity of postnasal drip, congestion, or sneezing. Seventy percent of patients treated with 42 micrograms of ipratropium bromide thought it had good or excellent effect on rhinorrhea (p less than 0.05 vs placebo); significantly more patients thought that it had improved the quality of life (p = 0.02). No changes occurred in nasal cytology, and no significant local or systemic adverse events occurred. These data indicate that ipratropium bromide significantly decreases the rhinorrhea of perennial allergic rhinitis.


Hemodynamic and sympathetic responses to human atrial natriuretic peptide infusion in patients with cirrhosis.

To determine the potential usefulness of atrial natriuretic peptide (ANP) in patients with cirrhosis, we examined the effects of the infusion of a low dose of alpha-human ANP (alpha hANP, 25 ng.kg-1.min-1 for 30 min) on renal, splanchnic, systemic hemodynamics and sympathetic outflow in eight patients. Pulmonary arterial plasma ANP concentrations increased from 59 +/- 9 to 328 +/- 41 pg/ml (mean +/- S.E., p less than 0.05). Mean values of glomerular filtration rate and renal plasma flow were not significantly changed. Individual renal plasma flow responses differed from one patient to another. Renal plasma flow increased in two patients, decreased in three and did not change in the other patients. Renal plasma flow changes were correlated with basal renal plasma flow values (r = -0.938, p less than 0.05) but not with arterial pressure changes or renal vein plasma norepinephrine concentration changes. Azygos blood flow increased from 0.43 +/- 0.10 to 0.63 +/- 0.13 l/min (p less than 0.05) and the hepatic-venous pressure gradient decreased from 19.9 +/- 1.5 to 17.5 +/- 2.9 mmHg in post-infusion (p less than 0.05). Mean arterial pressure decreased significantly by 18% and cardiac output by 12%. Systemic vascular resistance and pulmonary arterial plasma norepinephrine concentrations were not significantly modified. Thus, in patients with cirrhosis, alpha hANP appears to have a direct vasodilating action on renal arterioles when basal renal vascular tone is high. In addition, although alpha hANP might exert a portal hypotensive action, alpha hANP induced arterial hypotension as a result of both low cardiac output and a lack of increased sympathetic vascular tone. The arterial hypotensive action may, thus, limit the therapeutic use of low doses of alpha hANP in cirrhotic patients.


Mice deprived of exogenous antigenic stimulation develop a normal repertoire of functional T cells.

The development of T cells and the selection of the TCR repertoire in the absence of exogenous antigenic stimulation were investigated. For this purpose germfree BALB/c mice fed an ultrafiltered solution of chemically defined low m.w. nutrients (GF-CD) were used. Previous studies on B cell development and differentiation in GF-CD mice have demonstrated a high reduction in the number of cells secreting Ig of the non-IgM isotypes but an Ig-VH gene usage and a B cell specificity repertoire that is substantially different from that observed in conventional adult mice and more closely resembles that of neonatal conventional mice. In contrast, the present comparison of the various lymphocyte populations in the thymus, lymph nodes, and spleen from GF-CD and conventional mice using flow cytometry analysis revealed no significant differences. Analysis of the TCR-V beta expression on both mature thymocytes and lymph node T cells showed a high degree of similarity between GF-CD and conventional mice. These findings indicate a marked difference in the influence of exogenous antigenic stimulation on the development of B and T cells. Additionally, development in an environment free of exogenous antigenic stimulation allows for full functional maturation of T cells to occur, because MLC showed that GF-CD splenic T cells could mount allogeneic responses in a way similar to T cells generated in a conventional environment. Most importantly, full Th cell function is generated, because activation of GF-CD spleen cells by cross-linking with mAb against CD3 resulted in the induction of cells secreting IFN-gamma and Ig of the non-IgM isotypes, which cannot be detected in GF-CD sera. These findings demonstrate that functional T and B cells develop in mice that have not been exposed to exogenous Ag, and that the TCR repertoire, in contrast to the B cell compartment, is predominantly shaped by endogenously expressed Ag.


Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.


Regulation of human monocyte cell-surface and soluble CD23 (Fc epsilon RII) by granulocyte-macrophage colony-stimulating factor and IL-3.

We have investigated the regulation of expression of cell-surface and soluble CD23 (sCD23) by purified human peripheral blood monocytes and in cultures of human whole blood. IL-3, IL-4, and GM-CSF were found to markedly enhance the expression of CD23 on the surface of elutriated monocytes and to increase levels of sCD23 in monocyte-culture supernatants. The induction of CD23 expression by monocytes was confirmed at the mRNA level by Northern blot analysis. The ability of GM-CSF, IL-3, or IL-4 to induce cell-surface CD23 on monocytes was inhibited by specific neutralizing antibodies to the corresponding cytokine. IL-3 and GM-CSF induced maximal surface CD23 expression on monocytes by 24 to 48 h, followed by a slight decline at 72 and 96 h. In contrast, IL-4 induced a progressive increase in monocyte CD23 expression that reached a maximum at approximately 72 h. IL-4, GM-CSF, and IFN-gamma increased both surface and soluble CD23 expression by the monocytic cell line U937, whereas IL-3 had no effect. The plasma from fresh human whole blood or nonstimulated whole blood cultured for 24 to 48 h contained detectable sCD23, and addition of IL-3, IL-4, or GM-CSF to these cultures resulted in increased levels of this molecule. Two-color flow cytometry revealed that IL-3, but not GM-CSF, also enhanced CD23 expression by B cells enriched from PBMC, although the effect of IL-3 was weak in comparison with that of IL-4. These findings may have important implications for the in vivo therapeutic use of these cytokines.


IL-4 reciprocally regulates IL-1 and IL-1 receptor antagonist expression in human monocytes.

Activation of human monocytes with LPS induces coordinate expression of a number of cytokine genes, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8. The T cell-derived lymphokine, IL-4, inhibits expression of these genes in monocytes, suggesting that it may be an important physiologic regulator of cytokine production. We have previously shown that IL-4 reduces steady state messenger RNA (mRNA) levels for IL-1 beta in human monocytes by decreasing both IL-1 beta transcription and the t1/2 of newly formed IL-1 beta mRNA transcripts. In the present study, we extend these findings to show that IL-4 similarly accelerates the turnover of IL-6 mRNA in LPS-stimulated monocytes. However, this inhibition of cytokine expression and dramatic increase in the decay rate of cytokine mRNA does not extend to all LPS-inducible genes because IL-4 treatment did not inhibit the expression or accelerate the turnover of mRNA for the IL-1 receptor antagonist (IL-1ra) in the same cells. Although IL-1 beta and IL-1Ra are both LPS-inducible genes, they displayed distinct temporal patterns of expression. Peak steady state mRNA levels for IL-1ra lagged significantly behind that of IL-1 beta, suggesting a possible endogenous mechanism for limiting IL-1 biologic activity. Furthermore, although IL-4 suppressed expression of both IL-1 beta and IL-6, it up-regulated synthesis of IL-1ra mRNA and protein. Thus, IL-4 inhibits production of the proinflammatory cytokine, IL-1 beta, while concomitantly enhancing synthesis of the IL-1ra in activated human monocytes.


Murine recombinant Fc gamma RIII, but not Fc gamma RII, trigger serotonin release in rat basophilic leukemia cells.

Murine Fc gamma RII and Fc gamma RIII have highly homologous extracellular domains, but unrelated transmembrane and intracytoplasmic (IC) domains. Murine Fc gamma RIIb1 and b2 are two isoforms of single-chain receptors which differ only by 47 aa in their IC domain. Murine Fc gamma RIII are composed of an IgG-binding alpha-chain, the intracellular portion of which is unrelated to that of Fc gamma RII, and of a homodimeric gamma-chain which also associates with Fc epsilon RI. Murine mast cells express Fc gamma RII, Fc gamma RIII, and Fc epsilon RI. They can be induced to degranulate by murine IgG immune complexes or by F(ab')2 fragments of the rat anti-murine Fc gamma RII/III mAb 2.4G2, complexed to mouse anti-rat (MAR) F(ab')2. In order to determine which murine Fc gamma R can activate mast cells, cDNA encoding murine Fc gamma RIIb1, Fc gamma RIIb2 or Fc gamma RIII alpha were stably transfected into RBL-2H3 cells. Murine Fc gamma RIII but not Fc gamma RIIb1 or Fc gamma RIIb2 induced serotonin release when aggregated by (2.4G2-MAR) F(ab')2 complexes. The respective roles of the IC domains of murine Fc gamma RIII subunits in signal transduction were investigated by stably transfecting cDNA encoding IC-deleted or chimeric murine Fc gamma R into RBL-2H3 cells. The substitution of the IC domain of murine Fc gamma RII for that of murine Fc gamma RIII gamma, but not that of murine Fc gamma RIII alpha, conferred the ability to trigger serotonin release. The deletion of IC sequences of the alpha subunit did not alter the ability of murine Fc gamma RIII to trigger serotonin release. It follows that 1) murine Fc gamma RIII, but not Fc gamma RII, can induce RBL cells to release serotonin, 2) the aggregation of the IC domain of the murine Fc gamma RIII gamma subunit is sufficient, but 3) the IC domain of the murine Fc gamma RIII alpha subunit is neither sufficient nor necessary for triggering serotonin release.


Expression of complement components of the alternative pathway by glioma cell lines.

Glioma cell lines express proteins of the complement alternative pathway, namely C3, factor B, factor H, and factor I. Secretion of these proteins was shown by a sensitive and specific ELISA. C3 and factor H were rapidly secreted by glioma cell line CB193 and reached a concentration of 140 ng/ml/10(6) cells after 72 h of culture. Factor B and factor I were secreted at a lower rate and reached concentrations of 25 and 15 ng/ml/10(6) cells, respectively. Western blot and immunoprecipitation experiments showed that secreted proteins were identical to the corresponding plasma proteins. For factor H, besides the well known 150-kDa species, an additional polypeptide of 45 kDa with factor H immunoreactivity was observed. This species corresponded to the N-terminal truncated form found in plasma. In preliminary experiments, we observed control of these syntheses by cytokines. IL-1 beta significantly increased C3 secretion, with no effect on factor H. Secretion of factor H was enhanced by IFN-gamma. These results show that a glioma cell line could be a useful tool to study complement biosynthesis by glial cells in humans.


Early recognition of a discordant xenogeneic organ by human circulating lymphocytes.

Cell-mediated immune mechanisms underlying discordant xenograft rejection are poorly characterized. In our study, using a human to rat xenogeneic ex vivo model, we show that a fraction of human lymphocytes, when perfused through the coronary system of a rat heart, rapidly and specifically adheres to the vascular endothelium and infiltrates the myocardium. Lymphocyte phenotypic analysis before and after perfusion, as well as the use of purified cell subpopulations, demonstrate preferential adhesion of CD3- CD16+ NK cells. NK cell adhesion occurs via xenoreactive antibody-dependent and -independent pathways, because the selective removal of human IgG from the perfusion buffer markedly reduces but does not completely abrogate NK cell sequestration. However, T lymphocytes are retained in the xenoorgan via an antibody-independent pathway, as assessed by the lack of influence of IgG removal. Leukocyte integrins appear to play a crucial role in mediating adhesion of both lymphocyte subsets, because the pretreatment of lymphocytes with anti-CD11a, anti-CD11b, and anti-CD18 antibodies markedly reduces their retention into the xenogeneic organ. Retained human lymphocytes mediate rapid and direct damage of the xenoorgan, as demonstrated by histologic and functional alterations of the endothelium, impaired vascular resistance and in vitro lysis of rat endothelial cells by human NK cells. Taken together, these findings suggest a role for cell-mediated mechanisms in the rapid recognition and rejection of vascularized xenografts.


Class I restricted CTL recognition of a soluble protein delivered by liposomes containing lipophilic polylysines.

CD8+ cytotoxic lymphocytes recognize peptides derived from endogenous antigens complexed with class I major histocompatibility complex while CD4+ helper cells recognize peptides from exogenous antigens bound to class II MHC molecules. A soluble protein can be introduced into the class I pathway of antigen processing and presentation using an appropriate vehicle to deliver the antigen into the cytosol. Cationic liposomes containing lipophilic polylysine readily form complexes with an anionic, soluble protein ovalbumin. Mouse thymoma EL4 cells incubated with such complexes can be sensitized for killing by OVA-specific CTL effector cells. This method of target sensitization by a soluble antigen is more sensitive than the osmotic loading method previously reported.


[Inhibitory effects of calphobindins on phospholipase A2 and phospholipase C].

Calphobindins (CPBs) I, II and III which we isolated and characterized from placenta belong to a family of calcium and phospholipid binding proteins represented by lipocortins known to inhibit phospholipase A2(PLA2). The amino acid sequence of three CPBs shares much homology with lipocortins I and II, so we investigated the functions of CPBs in affecting the enzymatic activities of PLA2 and also phospholipase C(PLC). In our experiments CPBs I, II and III showed an apparent dose-dependent inhibition of PLA2 and PLC activities, and all three proteins had a more potent inhibitory effect than lipocortins. The inhibition was overcome by high phospholipid substrate concentrations, for example, in the presence of 2.9 x 10(-9)M PLA2 and 2.6 x 10(-7)M CPB I, the inhibition decreased from 85 to 9% as phosphatidylethanolamine was increased from 10 to 100 microM. A similar result was observed in the case of PLC also. The evidence strongly suggests that the inhibitory effect of CPBI results from binding of the proteins to the phospholipid substrate rather than from a direct action on the phospholipase itself.


Induction of gamma/delta T cells in murine salmonellosis by an avirulent but not by a virulent strain of Salmonella choleraesuis.

To elucidate the relationship between the virulence of intracellular bacterium and its ability to induce gamma/delta T cells in the host during infection, we examined the differences in appearance of gamma/delta T cells in mice infected with Salmonella choleraesuis virulent strain RF-1 carrying a virulence plasmid of 50 kb, and with avirulent strain 31N-1 cured of the 50-kb plasmid. The number of gamma/delta T cells in the peritoneal cavity was increased to a significant level on day 3 after an intraperitoneal infection with a sublethal dose (5 x 10(4) colony-forming units) of avirulent strain 31N-1. On the other hand, no increase in the number of gamma/delta T cells was evident in the peritoneal cavity at any stage after infections with various doses of virulent strain RF-1, although the numbers of the bacteria were drastically increased. Similar to that seen in the peritoneal cavity, the number of gamma/delta T cells in the liver was significantly increased after an intraperitoneal infection with avirulent strain 31N-1 but not with virulent strain RF-1. The early appearing gamma/delta T cells during salmonellosis with avirulent stain 31N-1, which preferentially used V gamma 1/V delta 6, showed blastogenesis in response to purified protein derivative (PPD) derived from Mycobacterium tuberculosis. The gamma/delta T cells also responded to the peritoneal adherent cells in mice infected with avirulent strain 31N-1 6 d previously, which expressed a high level of endogenous heat-shock protein (hsp) homologous to the mycobacterial 65-kD hsp. The expression of the hsp, however, was not prominent in the adherent cells in mice infected with virulent strain RF-1. These results suggest that the gamma/delta T cells specific for PPD may play important roles in host defense against murine salmonellosis, and that the virulence of Salmonella may be inversely correlated with its ability to induce endogenous hsp in the infected macrophages, which in turn stimulate the gamma/delta T cells in the host during salmonellosis.


Expression of a transgenic T cell receptor beta chain enhances collagen-induced arthritis.

SWR/J transgenic (tg) mice were generated expressing the TCR beta chain derived from an anticollagen type II (CII) arthritogenic T cell clone. The SWR/J strain was selected because it is resistant to collagen-induced arthritis (CIA) and lacks the V beta gene segment used by the T cell clone. Expression of the tg beta chain on all thymocytes and peripheral lymph node T cells led to a more efficient anti-CII immune response, but did not confer CIA susceptibility to SWR/J mice. Nevertheless, this tg beta chain enhanced predisposition to CIA as (DBA/1 x SWR) F1 beta tg mice were more susceptible than normal F1 littermates. Our results demonstrate that the expression of the tg beta chain contributes to CIA susceptibility, but by itself it is not sufficient to overcome CIA resistance in the SWR/J strain.


Demonstration of a second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into fluorescent liposomes.

Recombinant full-length human CD23 has been incorporated into fluorescent liposomes to demonstrate the existence of a ligand for CD23 that is different from the previously known ligand, immunoglobulin E (IgE). The novel ligand for CD23 is expressed on subsets of normal T cells and B cells as well as on some myeloma cell lines. The interaction of full-length CD23 with its ligand is specifically inhibited by anti-CD23 monoclonal antibodies and by IgE, and it is Ca2+ dependent. Moreover, tunicamycin treatment of a CD23-binding cell line, RPMI 8226, significantly reduced the binding of CD23 incorporated into fluorescent liposomes, and a sugar, fucose-1-phosphate, was found to inhibit CD23-liposome binding to RPMI 8226 cells, suggesting the contribution of sugar structures on the CD23 ligand. In addition, CD23-transfected COS cells were shown to form specific conjugates with the cell line RPMI 8226. These data demonstrate that CD23 interacts with a ligand, which is different from IgE, and that CD23 can be considered as a new surface adhesion molecule involved in cell-cell interactions.


Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as Fc gamma RII and Fc gamma RIII.

In addition to their well characterized high affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) mast cells have long been suspected to express undefined Fc receptors capable of binding IgE with low affinity. In this paper, we show that Fc gamma RII and Fc gamma RIII, but not Mac-2, on mouse mast cells and macrophages bind IgE-immune complexes. This binding is efficiently competed by 2.4G2, a monoclonal antibody against the extracellular homologous region of both Fc gamma RII and Fc gamma RIII. Furthermore, IgE-immune complexes bind specifically to Fc gamma RII or Fc gamma RIII transfected into COS-7 cells. The association constants of IgE binding estimated from competition experiments are about 3.1 x 10(5) M-1 for Fc gamma RII, and 4.8 x 10(5) M-1 for Fc gamma RIII. Engagement of Fc gamma RII and Fc gamma RIII with IgE-immune complexes (after blocking access to Fc epsilon RI) or with IgG-immune complexes triggers C57.1 mouse mast cells to release serotonin. This release is inhibited by 2.4G2, and at maximum, reaches 30-40% of the intracellular content, about half of the maximal release (60-80%) obtained after Fc epsilon RI engagement. These data demonstrate that mouse Fc gamma RII and Fc gamma RIII are not isotype specific, and that the binding of IgE-immune complexes to these receptors induces cell activation.


Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.


Differentiation of CD3-4-8- thymocytes in short-term thymic stromal cell culture.

We have investigated the ability of a heterogeneous thymic stromal cell (HTSC) culture system to promote in vitro differentiation of CD3-4-8- thymocytes. Culture of purified murine CD3-4-8- thymocytes on HTSC for 1 d resulted in the appearance of CD4+8+ cells, which did not occur when the sorted cells were maintained in medium alone. It is remarkable that when the culture period was extended to 2 d, CD3-4-8- progenitors differentiated further to CD4+8- and CD4-8+ cells, which also expressed high levels of TCR-CD3. This rapid differentiation on stroma in vitro appears to outpace parallel development in vivo. The differentiation potential of a subset of CD3-4-8- thymocytes that express high levels of a marker of normal and neoplastic thymic progenitors, the 1C11 antigen, was examined next. 1C11hiCD3-4-8- cells also gave rise to CD4-8+ and CD4+8+ populations after 1 d of culture on HTSC. Extending the culture period to 2 d resulted in a significant percentage of CD3-expressing cells that were CD4+8+, CD4+8- and CD4-8+ cells. These results suggest that in the in vitro HTSC culture system, various subsets of immature thymocytes can differentiate into all the mature phenotypes of cells normally found in the adult mouse thymus. This may provide a novel and rapid assay for thymic progenitors.


A fail-safe mechanism for maintaining self-tolerance.

Using cytotoxic T lymphocyte (CTL) responses to the class I histocompatibility antigen Qa1 and to the minor histocompatibility antigen H-Y, we show that the immune system maintains a peripheral screening process that is able to tolerize a wide variety of potentially autoimmune CTL. The critical factor is the presence or absence of specific T helper cells. If T help is available, CTL precursors that recognize antigen are activated. In the absence of help, they are tolerized. Thus, T helper cells are guardians of peripheral tolerance in CTL.


Human neutrophils produce high levels of the interleukin 1 receptor antagonist in response to granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha.

Neutrophils, an abundant cell type at sites of inflammation, have the ability to produce a number of cytokines, including interleukin 1 (IL-1), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha). In this study, we have examined the ability of human neutrophils to produce the IL-1 receptor antagonist (IL-1Ra), a 17-23-kD protein recently isolated and cloned from macrophages. Since IL-1Ra has been shown to inhibit both the in vitro and in vivo effects of IL-1, its production by large numbers of tissue-invading neutrophils might provide a mechanism by which the effects of IL-1 are regulated in inflammation. Using antibodies that are specific for IL-1Ra and a cDNA probe encoding for this protein, we were able to show that neutrophils constitutively produce IL-1Ra. However, after activation by GM-CSF and TNF-alpha, IL-1Ra was secreted into the extracellular milieu where it constituted the major de novo synthesized product of activated neutrophils. None of a large array of other potent neutrophil agonists were found to affect the production of IL-1Ra by neutrophils. Quantitative measurements by enzyme-linked immunosorbent assay revealed that intracellular IL-1Ra is in eightfold excess of the amount secreted in supernatants when studying nonactivated neutrophils. However, in GM-CSF- and TNF-alpha-activated cells, this difference was reduced to values between four- and fivefold, as virtually all of the de novo synthesized IL-1Ra was secreted. In activated cells, the intracellular content of IL-1Ra was found to be in the 2-2.5-ng/ml range per 10(6) neutrophils, whereas levels reached the 0.5-ng/ml range in supernatants. This would imply that IL-1Ra is produced in excess of IL-1 by a factor of at least 100, an observation that is in agreement with the reported amounts of IL-1Ra needed to inhibit the proinflammatory effects of IL-1. Neutrophils isolated from an inflammatory milieu, the synovial fluid of patients with rheumatoid arthritis, were found to respond to GM-CSF and TNF-alpha in terms of IL-1Ra synthesis, indicating that the in vitro observations made in this study are likely to occur in an inflammatory setting in vivo.


A quantitative autoradiographic study of 125I atrial natriuretic factor in the heart of a teleost fish (Conger conger).

Quantitative receptor autoradiographic study of 125I-atrial natriuretic peptide factor (ANF) in the heart of a teleost fish Conger conger has shown that a heterogenous distribution of 125I-ANF binding exists in the different cardiac regions. Elevated ANF binding densities (3,790 fmol/mg protein) were encountered in the innermost layer (tunica intima) of the bulbus arteriosus while lower binding levels (293-403 fmol/mg protein) were revealed in atrium and ventricle. In order to determine 125I-ANF binding characteristics (KD, Bmax) in the above cardiac sites, saturation binding assays were carried out. The results show that low 125I-ANF KD values (28.8-52.6 pM) were found in the atrium and in the bulbus arteriosus with respect to the higher KD values (373 pM) of the ventricle. The number of binding sites were respectively 632 and 1,279 fmol/mg protein for the atrium and the ventricle, while a substantially elevated Bmax of 7,235 fmol/mg protein was found for the bulbus arteriosus. These results may furnish some insights concerning ANF receptor binding activity and its putative regulatory role of different cardiac functions.


Fourier transform infrared analyses of some particulate drug mixtures using a diamond anvil cell with a beam condenser and an infrared microscope.

Although the diamond anvil cell (DAC) has been used in many forensic science laboratories for the analysis of trace evidence, few applications of this technique for the analysis of controlled substances have been reported. This may be due to both an unfamiliarity on the part of forensic drug chemists with this accessory and the nature and quality of spectra that result from use of a DAC on a dispersive instrument. Along with low energy throughput, which results in relatively high noise levels, strong broad diamond absorptions occur. With the use of a Fourier transform infrared instrument, these do not present a problem and nanogram quantities of materials can be analyzed when the DAC is used with an infrared microscope. Since single crystals can be sampled with the DAC, simple physical separations (involving particle-picking) can be used in certain cases to isolate drugs from particulate mixtures for infrared analysis. This method is especially useful for some "difficult" mixtures and residues, and several examples of such analyses involving samples of forensic science interest are presented.


Visualization method for fingerprints on skin by impression on a polyethylene terephthalate (PET) semirigid sheet.

Based on the phenomenon that static electricity attracts dust, a polyethylene terephthalate (PET) semirigid sheet coated with printing ink was used to visualize impressions of fingerprints on the skin of living and dead bodies. Compared with visualization methods for perspiration fingerprints, this method recovers better images for a longer time after the fingerprint has been deposited on skin. Fingerprints transferred to the PET sheet are photographed with sidelighting using an ordinary light source. For fingerprints that yield inadequate contrast, an argon-ion laser can be used to improve the contrast.


Protein C, protein S and C4b-binding protein in neonatal severe infection and septic shock.

We have studied the behaviour of total protein S, free protein S, protein C and C4b-binding protein fifteen neonates with severe infections, eight with septic shock and in a group of ten healthy newborns. Protein C was decreased in shock and septic patients, but only the shock group showed significant differences compared to normal neonates. Total protein S was normal in both groups of patients, although free protein S had significantly lower values in shock and nonshock infants. C4b-binding protein was higher than normal in septic and shock patients compared to the control group. Decreased values of protein C and free protein S can be explained by the activation of coagulation and their subsequent consumption. On the other hand, the increased levels of C4b-binding protein can affect the distribution of protein S in plasma, producing a shift in protein S to the complexed inactive form. These findings can contribute to an increased risk of microthrombosis during neonatal sepsis.


Sensitization to the toxic effects of cocaine in mice is associated with the regulation of N-methyl-D-aspartate receptors in the cortex.

Repeated exposure to cocaine results in sensitization to many of the behavioral effects of the drug. The present study was undertaken to examine the role of the N-methyl-D-aspartate (NMDA) type of glutamate receptors in the development of sensitization to the convulsive and lethal effects of cocaine in Swiss Webster mice. Repeated administration of subconvulsant doses of cocaine (45 mg/kg for 7 days) produced a progressive increase in the convulsive responsiveness to the drug. This phenomenon was accompanied by an increase in lethality rate after the 5th day of the treatment. Pretreatment with the noncompetitive NMDA receptor antagonist, MK-801 (5-methyl-10,11-dihydro-5H-dibenzo[a,d]- cyclohepten-5,10-imine) abolished completely the development of sensitization to cocaine-induced seizures and lethality. In addition, MK-801 attenuated cocaine-induced loss in animals body weight after 7 days of drug treatment. The lethal effects of acute administration of increasing doses of cocaine were also reduced by pretreatment with MK-801. In vitro receptor binding experiments demonstrated an increase (139% of control) in the number of NMDA receptors, labeled with the competitive NMDA receptor antagonist [3H]CGP 39653 ([3H]-2-amino-4-propyl-5-phosphono-3-pentenoic acid), in cortical membranes derived from the mice treated for 7 days with cocaine (45 mk/kg). In agreement with the latter finding, binding of [3H]MK-801 to the phencyclidine/NMDA site in cortical membranes of cocaine-treated mice was more sensitive to the stimulatory effect of glutamate compared to control (saline treatment).(ABSTRACT TRUNCATED AT 250 WORDS)


Mast cell mediators regulate vascular permeability changes in Arthus reaction.

Plasma exudation characterizes the early phase of acute inflammation. The possible role of mast cells and their mediators in this event in immune complex-induced injury was studied. Dye exudation was assessed from 5 min to 2 hr after initiating reverse passive Arthus reaction in mast cell-deficient mice, WBB6F1-W/Wv (W/Wv), and their normal congenic controls, WBB6F1-+/+ (+/+). The response to antibody (10, 30 and 100 micrograms/site, i.d.) was dose- and time-dependent in both groups of mice. At the lower doses of antibody, 10 and 30 micrograms/site, exudation was significantly less (30% and 40%, respectively) in W/Wv as compared to +/+ mice between 15 to 45 min. With 100 micrograms of antibody/site, significant differences between W/Wv and +/+ mice were noted only at 15 and 30 min. The deficit in permeability changes in W/Wv mice was reversed by local mast cell reconstitution. In +/+ mice, pyrilamine and methysergide pretreatment reduced vascular permeability to the same extent by 70, 60 and 35% when stimulated for 30 min with 10, 30 and 100 micrograms of antibody/site, respectively. An equivalent inhibition was observed with the 5-lipoxygenase inhibitor A-63162. None of the inhibitors decreased plasma permeation in W/Wv mice. These results indicate that the mast cell mediators histamine and serotonin regulate vascular permeability early during an immune complex-mediated inflammation. The data also suggest the involvement of leukotrienes and the importance of mast cells in their synthesis. The profile of inhibition in +/+ mice agrees well with the difference in exudation observed between normal and mast cell-deficient mice.


Effect of short-term treatment with gastrin and related peptides on gastrointestinal histamine H2-receptors.

The effects of short-term, 7-day, treatment with synthetic 15-leucine human gastrin I, pentagastrin or sulfated cholecystokinin-8 on the activity of histamine (HA)-stimulated adenylate cyclase in membranes isolated from guinea pig gastric mucosa and H2-receptor-mediated contractions of isolated ilea were evaluated. Treatment with each of the peptides produced a decrease in the maximal rate of HA-stimulated adenylate cyclase. The decreases in the maximal rate occurred without any effect on the potency of HA or any effect on basal rates of activity. In animals treated with pentagastrin, but not with cholecystokinin octapeptide sulfate, the contractile activity of dimaprit, a selective H2-agonist, was decreased. In animals treated with pentagastrin, the contractile actions of pentagastrin on isolated ileal preparations were increased. A 7-day treatment with the H2-antagonist, tiotidine, did not alter the potency of or the maximal response for HA-stimulated adenylate cyclase activity. Co-treatment with tiotidine prevented the effects of pentagastrin on gastric mucosal HA-stimulated adenylate cyclase. Treatment with pentagastrin did not alter the sensitivity of the gastric mucosal H2-receptor to inhibition by tiotidine. The effects of treatment with gastrin on NaF-stimulated adenylate cyclase activity also were determined. Treatment with gastrin did not alter the actions of NaF, suggesting that the coupling between the Gs subunit and the catalytic subunit of adenylate cyclase was not altered.(ABSTRACT TRUNCATED AT 250 WORDS)


7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid: a novel high-affinity radiolabeled antagonist for platelet thromboxane A2/prostaglandin H2 receptors.

A high-affinity thromboxane (TX)A2/prostaglandin (PG) H2 receptor antagonist, I-SAP [7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid] and its radiolabeled analog [125I]SAP (Mais et al., 1991) are characterized in the present study. I-SAP antagonized I-BOP ([1S-(1 alpha, 2 beta(5Z),3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2y l]-5'heptenoic acid) and U46619 [15S-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5Z,13E-dienoic acid)], two different TXA2/PGH2 mimetics, induced aggregation of washed human platelets in a similar manner (pA2 of 8.11 +/- 0.09, Kd = 7.8 nM, n = 3; pA2 = 8.01 +/- 0.05, Kd = 9.7 nM, n = 8, respectively). I-SAP also had agonistic activity, producing platelet shape change (EC50 = 9.7 nM +/- 0.6 nM at pH 7.4, n = 3) which was blocked by pretreatment of platelets with SQ29548 ([1S-(1 alpha,2 beta(5Z),3 beta,4 alpha)]-7-[3-[[2- [(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept- 2-yl]-5-heptenoic acid), a TXA2/PGH2 receptor antagonist. Radioligand binding studies were performed with [125I]SAP using washed human platelets. Competition of three agonists and four antagonists for binding with [125I]SAP was determined. The compounds showed the appropriate rank order potencies, including stereoselective competition by a pair of stereoisomeric antagonists. In washed human platelets, the Kd for I-SAP was 468 +/- 49 pM and the maximum binding (Bmax) was 2057 +/- 156 sites/platelet at pH 7.4 (n = 6). The Bmax was significantly increased 49% to 3072 +/- 205 sites/platelet at pH 6.5 (P less than .01 but the Kd was unchanged (490 +/- 18 pM, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of indole-2-carboxylate derivatives as antagonists of N-methyl-D-aspartate receptor activity at the associated glycine recognition site.

We have synthesized a series of indole-2-carboxylate derivatives and, with the use of radioligand binding, electrophysiological techniques and an in vivo transient bilateral carotid occlusion model of ischemic damage known to be sensitive to NMDA antagonists, have evaluated the indole-2-carboxylate derivatives ability to inhibit N-methyl-D-aspartate (NMDA) receptor activity through the associated glycine modulatory site. By using [3H]glycine to label this modulatory site, we found that the compounds with the highest affinity (Ki less than 1 microM) contained a chloro group at position C-6 and a polar, hydrogen-bond-accepting group at position C-3 of the indole ring. When these compounds were tested for their ability to modulate [3H]MK-801 [(+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclophepten-5,10- imine maleate) binding, a functional assessment of NMDA receptor activation, binding was inhibited, indicative of NMDA receptor antagonist character. Schild regression analysis indicated that this antagonism was competitive with glycine. Next, several of these indole-2-carboxylate derivatives were analyzed electrophysiologically in rat cortex mRNA-injected Xenopus oocytes shown to express a functional NMDA receptor channel complex. These compounds inhibited NMDA receptor activity in a manner noncompetitive with NMDA. They also produced a parallel right-ward shift in the glycine dose response for potentiation of the NMDA responses in the oocytes and thus provided further evidence for a competitive interaction at the glycine site. Finally, in vivo transient bilateral carotid artery occlusion experiments revealed that these compounds were capable of reducing the damage typically associated with an ischemic insult in Mongolian gerbil hippocampal neurons.


Actions of a 5-lipoxygenase inhibitor, Sch 40120, on acute inflammatory responses.

Sch 40120 (10-(3-chlorophenyl)-6,8,9,10-tetrahydrobenzo[b] [1,8]naphthyridin-5(7H)-one) is an inhibitor of the 5-lipoxygenase enzyme in rat neutrophils, human neutrophils and the the MC9 murine mast cell clone with IC50 values of 8, 4 and 7 microM, respectively. The drug was examined for its effects on acute inflammatory responses in the paw and pleural cavity of rats. The drug suppressed paw inflammation triggered by a reverse passive Arthus reaction or a subplantar injection of the polysaccharide carrageenan with p.o. ED50 values of 0.2 and 1.5 mg/kg, respectively. In reverse passive Arthus reaction and carrageenan pleurisy models, Sch 40120 was found to suppress both the cellular and fluid components of the acute inflammation. The p.o. ED50 values for inhibition of cells and fluid in pleurisy models were in the range of 0.1 to 0.7 mg/kg. When applied locally to the ears of mice, the drug blocked an arachidonic acid-induced and leukotriene-mediated ear inflammation with an ED50 of 0.072 mg/ear. These findings suggest that Sch 40120 is a potent anti-inflammatory agent that may be particularly useful in the treatment of inflammatory diseases such as psoriasis in which leukotrienes appear to be major mediators of the pathological symptoms that characterize the disease state.


In vitro characterization of a novel TXA2/PGH2 receptor ligand (S-145) in platelets and vascular and airway smooth muscle.

The stereoisomers of S-145, a novel thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor ligand, were compared to TXA2/PGH2 receptor antagonists, SQ29548 and BM13505 in guinea pig platelets, aortas and trachea. Equilibrium binding assays in platelets yielded Kd values (nanomolar) for (+)-S-145 (0.57 +/- 0.04), (-)-S-145 (9.2 +/- 1.3), SQ29548 (11.1 +/- 0.70) and BM13505 (118 +/- 16). In aortas, the corresponding Kb values (nanomolar) were (0.014 +/- 0.002), (1.90 +/- 0.31), (16.8 +/- 3.3) and (142 +/- 29), respectively, whereas in trachea, the Kd values (nanomolar) were (0.019 +/- 0.004), (1.12 +/- 0.18), (1.94 +/- 0.30) and (18.99 +/- 2.59), respectively. S-145 stereoisomers elicited platelet shape change stereoselectively that was characterized by EC50 values 8 to 16-fold higher than the EC50 values for these ligands to block aggregation induced by TXA2/PGH2 mimetic, U44069. S-145 (+)- and (-)-isomers stereoselectively induced transient aortic contraction at concentrations 214,000- and 16,000-fold higher, respectively, than the corresponding Kb values in this tissue. S-145-induced platelet shape change and aortic contraction were inhibitable by low concentrations of SQ29548. We postulate that S-145 may elicit partial agonist activity in platelets and aorta via lower affinity for the active than inactive state of the TXA2/PGH2 receptor in those tissues. S-145 had no agonist activity in isolated trachea possibly indicating different TXA2/PGH2 recognition sites in aorta and trachea or a smaller preligand ratio of active to inactive TXA2/PGH2 receptors in trachea than in aorta.