Hemophilic chronic synovitis: therapy of hemarthrosis using endovascular embolization of knee and elbow arteries.

PURPOSE: Congenital hemophilia is a hereditary bleeding disorder that affects 1 in 5,000 males and is characterized by repetitive musculoskeletal bleeding episodes. Selective embolization of the knee and elbow arteries can prevent bleeding episodes. To evaluate the long-term efficacy of these procedures, we assessed the outcomes of 30 procedures performed in our center. METHODS: We performed 30 procedures in 27 hemophilic patients, including 23 knee, and 7 elbow procedures. To evaluate the efficacy of selective embolization of knee and elbow arteries in people with hemophilia, we analyzed the number of bleeding episodes during 12 months before the procedure compared with the amount of episodes that occurred 3, 6, and 12 months after embolization. RESULTS: Twenty-nine of 30 procedures were classified as successful. The median of 1.25 episodes per month (range 0-3) observed before the procedure was reduced to 0 (range 0-1.67; p < 0.001) at 3 months, 0.17 (range 0-1.67; p < 0.001) at 6 months, and 0.33 (range 0-1.67; p = 0.024) at 12 months. Three patients remained free of bleeding events for more than 6 months. Additionally, after the procedure there was a significant reduction in factor FVIII usage that sustained up to 12 months after the procedures. No serious adverse events were observed. CONCLUSIONS: Selective angiographic embolization of knee and elbow arteries is a feasible procedure that can prevent repetitive bleedings, which would translate in better joint outcomes for these patients.

Elective orthopaedic surgery for haemophilia patients with inhibitors: single centre experience of 40 procedures and review of the literature.

With the introduction of safe and effective factor VIII/IX-bypassing agents--recombinant activated factor VII (rFVIIa) and plasma-derived activated prothrombin complex concentrates (pd-APCC)--elective orthopaedic surgery (EOS) is a viable option for haemophilia patients with inhibitors. We report a series of patients with haemophilia and inhibitors undergoing EOS between 1997 and 2008 using bypassing agents to provide haemostatic cover. All inhibitor patients undergoing EOS and receiving rFVIIa, plasma-derived prothrombin complex concentrates (pd-PCC) or pd-APCC as haemostatic cover were included. Patients were operated on by the same surgeon and were managed by the same haemophilia treatment centre. Forty procedures (25 minor and 15 major) were conducted in 18 patients. Twenty-one minor cases were covered using rFVIIa, three with pd-PCC, and one with pd-APCC; all major cases were covered using rFVIIa. Bleeding was no greater than expected compared with a non-haemophilic population in all 25 minor procedures. In the major procedure group, there was no excessive bleeding in 40% of cases (6/15) and bleeding completely stopped in response to rFVIIa. For the remaining nine cases, bleeding response to rFVIIa was described as 'markedly decreased' or 'decreased' in 4/15 cases and 'unchanged' in 5/15 cases. Overall, efficacy of rFVIIa, based on final patient outcome, was 85%. One death occurred as a result of sepsis secondary to necrotizing fasciitis. Good control of haemostasis can be achieved with bypassing agents in haemophilia patients with inhibitors undergoing minor EOS; rFVIIa was used as an effective bypassing agent, enabling EOS in patients undergoing minor and major procedures.

Sialic acid content in erythrocyte membranes from pregnant women affected by gestational diabetes.

Sialic acid (SA) content, membrane fluidity, and Na(+)/K(+)-adenosine triphosphatase (ATPase) activity were determined in erythrocyte membrane from 10 nonpregnant women (HNPW), 16 pregnant women affected by gestational diabetes mellitus (GDM), and 25 healthy pregnant women (HPW). In GDM patients the membrane erythrocyte SA content was significantly increased compared with HNPW and membrane fluidity was significantly increased in comparison with HPW. Erythrocyte membrane Na(+)/K(+)-ATPase activity was significantly reduced in GDM patients compared both to HNPW and to HPW subjects. A significant inverse correlation was found between 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) anisotropy and erythrocyte membrane SA content in HNPW and in HPW, while this significant correlation was not observed in GDM. The present results indicate that in comparison with normal pregnancy GDM is characterized by deep alterations of the erythrocyte plasma membrane physicochemical properties (increased fluidity) and functional activities (reduced Na(+)/K(+)-ATPase activity). These modifications might be at the basis of the altered blood viscosity and placental perfusion observed under such conditions. Moreover, these results show that in physiological pregnancy and in the nonpregnant state, the erythrocyte surface membrane fluidity is inversely correlated with SA content, while in GDM there is an unbalance of this relation, which might be associated with the microcirculatory abnormality present in this disease.

Reduced susceptibility to peroxidation of erythrocyte plasma membranes from centenarians.

The plasma membrane composition affects intracellular processes and the cellular susceptibility to free radical attack, which has been associated with the impairment of cellular functions occurring during senescence. The study of the modifications of the plasma membrane in centenarians might elucidate the biological mechanisms at the basis of longevity and successful aging. The work was performed in 190 subjects, divided into five groups according to the age range: (1) 21-40 years (n=25); (2) 41-60 years (n=30); (3) 61-80 years (n=30); (4) 81-99 years (n=50); and (5) centenarians (> or = 100 years) (n=55). The following determinations were performed on erythrocyte membranes: (i) the lipid peroxide level (Lp) evaluated as malondialdehyde content; (ii) susceptibility to in vitro oxidation evaluated as difference in the content of thiobarbituric acid-reactive substances before and after phenylhydrazine addition; (iii) unsaturated/saturated fatty acid ratio and individual polyunsaturated fatty acid composition measured by gas chromatography; and (iv) fluidity studied by means of the anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Erythrocyte membranes from centenarians showed: (i) decreased basal lipid peroxide levels and reduced susceptibility to peroxidation in comparison with elderly subjects; (ii) increased unsaturated/saturated fatty acid ratio in comparison with every other age group; (iii) higher levels of eicosapentaenoic and docosahexaenoic acid and reduced content of linoleic and arachidonic acid in comparison with elderly subjects; and (iv) decreased anisotropy of TMA-DPH, i.e. higher fluidity compared with all the other age groups. In conclusion, the present work demonstrates that erythrocyte membranes from centenarians show some distinct features in comparison with elderly subjects that might act in a protective way against injuries.

Homocysteine-induced inhibition of nitric oxide production in platelets: a study on healthy and diabetic subjects.

AIMS/HYPOTHESIS: The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects. METHODS: The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 micromol/l homocysteine. Stimulation experiments were done in vitro by the addition of alpha-thrombin (0.2 U/ml). RESULTS: Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects. CONCLUSION/INTERPRETATION: Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation.

Decreased nitric oxide levels and increased calcium content in platelets of hypertensive patients.

Decreased levels of nitric oxide (NO) may contribute to impaired endothelium-dependent vasodilatation in essential hypertension. Moreover, in hypertension, major platelets aggregation and endothelial adhesion, and increased atherogenetic risks are also present. Nitric oxide produced by platelet NO synthase, which is similar to endothelial NO synthase, inhibits platelets aggregation by increasing cytoplasmic cyclic GMP levels and contributes in a major way to the antithrombogenic properties of endothelium. The aim of this study was to investigate platelet NO production and cytosolic Ca2+ levels in patients with essential hypertension and in healthy subjects. We studied NO production in 36 subjects (21 patients had essential hypertension and 15 subjects were normotensive); NO synthase activity was evaluated by measuring nitrite levels by the Griess reaction in the supernatant of sonicated platelets. Cytosolic Ca2+ levels were measured in intact platelets using the fluorescent probe Fura 2-AM. Nitric oxide levels in platelets were found higher in normotensive than in hypertensive patients (P < .0001). Nitric oxide levels in hypertensive women were significantly higher than in hypertensive men (P < .001). Hypertensive women and men had lower levels of nitrite than normotensive women and men (P < .001 and P < .002, respectively). Platelet cytosolic Ca2+ levels were higher in hypertensive patients than in normotensive subjects (P < .001). An inverse correlation was found between platelet cytosolic Ca2+ and NO levels (r = 0.74, P < .002). These data confirm the link between hypertension and altered platelets function and suggest a role for NO in cardiovascular events. Moreover, the higher levels of nitric oxide in child-bearing age women than in men further support the protective effect of estrogens on cardiovascular diseases.

Serotonergic nerve fibers in the primary olfactory pathway of the larval sea lamprey, Petromyzon marinus.

In this study, serotonin (5-hydroxytryptamine; 5HT)-immunoreactive (5HT-IR) neuronal fibers were identified in the primary olfactory pathway of the sea lamprey. These neurons are likely part of a nonolfactory neural system that innervates the olfactory sac. Cell bodies with 5HT immunoreactivity predominated in the lamina propria of the rostral portion of the nasal cavity and were less prevalent adjacent to the olfactory epithelium. The 5HT-IR fibers were parallel to axons of the olfactory receptor neurons in the lamina propria of the olfactory mucosa and in the olfactory nerve. Serotonergic fibers crossed from the olfactory nerve into the olfactory bulb or branched in the caudal portion of the olfactory nerve and terminated at the junction of the olfactory nerve with the olfactory bulb. In the dorsal olfactory bulb, 5HT-IR fibers coursed along the layer of olfactory fibers. Throughout the layer with glomeruli and mitral cells, 5HT-IR fibers were seen along the border of glomerular units. Experimental lesion of the olfactory nerve was used to determine the origin of 5HT-IR fibers rostral to the olfactory bulb. The loss of these fibers and their reappearance during outgrowth of olfactory receptor neurons inferred that they emanate from the cell bodies in the olfactory sac. The results from this study suggest that axons of olfactory receptor neurons in larval lampreys receive modulation by 5HT from these neuronal fibers.

Modifications induced by LDL from type 1 diabetic patients on endothelial cells obtained from human umbilical vein.

The aim of the present work was to analyze the effect of LDL obtained from type 1 diabetic patients in good metabolic control on human umbilical vein endothelial cells (HUVECs) after a short incubation period to detect possible atherogenic modifications of endothelial properties. Cultured HUVECs were incubated for 3 h with culture medium alone (control HUVEC), with native LDL from 12 healthy men (control LDL), or with native LDL from 12 type 1 diabetic men (type 1 LDL) (100 pg/ml). After the incubation, the following parameters were evaluated: nitric oxide synthase (NOS) activity, cytoplasmic Ca2+ levels, Na+-K+-ATPase activity, plasma membrane fluidity determined by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and plasma membrane conjugated diene (CD) content. The same experiments were repeated after bradykinin stimulation or in the presence of the antioxidant butylated hydroxytoluene (BHT), and nitric oxide (NO) production in intact HUVECs was also evaluated. HUVECs incubated with control LDL in comparison with control HUVECs showed a decreased fluidity of the membrane surface evaluated by TMA-DPH and a higher CD content. These alterations were prevented by the presence of BHT. HUVECs incubated with type 1 LDL in comparison with both control HUVECs and cells incubated with control LDL showed 1) increased NOS and Na+-K+-ATPase activity, cytoplasmic Ca2+ levels, and CD content, and 2) decreased fluidity of the membrane surface evaluated by TMA-DPH. These modifications were blunted--but not abolished--by the presence of BHT. After bradykinin stimulation either in the absence or in the presence of BHT, both cytoplasmic Ca2+ levels and NO production were increased in control HUVECs and in HUVECs incubated with control LDL, while a reduced response was observed in HUVECs incubated with type 1 LDL. The alterations observed in the endothelial function after the cell-LDL interaction might play a central role in the atherogenic process in diabetes.

A study on human umbilical cord endothelial cells: functional modifications induced by plasma from insulin-dependent diabetes mellitus patients.

The aim of the present study was to evaluate the action of plasma from insulin-dependent diabetic (IDDM) pregnant women on nitric oxide synthase (NOS) activity in cultured human umbilical vein endothelial cells (HUVECs). We also studied the effect of the plasma on cytosolic calcium and on Na+/K+-adenosine triphosphatase (ATPase) activity. Dynamic fluorescence studies of membrane fluidity were contemporarily performed to detect a direct effect of plasma on the endothelial cell membrane. We observed a significant increase in NOS activity, intracellular calcium, and Na+/K+-ATPase activity in cultured HUVECs exposed to IDDM plasma. Our dynamic fluorescence study showed a different microenvironmental organization of the cellular membrane after incubation with plasma from IDDM pregnant women, with a marked decrease in microheterogeneity as evaluated in terms of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) lifetime distribution width. The present investigation suggests that plasma from IDDM pregnant women can cause a generalized disturbance in the function of endothelial cells cultured from healthy subjects. Such a modification might play a central role in the pathogenesis of the vascular complications of the disease.

Evidence for iNOS-dependent peroxynitrite production in diabetic platelets.

AIMS/HYPOTHESIS: The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase. METHODS: Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting. RESULTS: In the presence of extracellular L-arginine (100 micromol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of L-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase. CONCLUSION/INTERPRETATION: Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes.

[Optimization of the pertussis vaccine production process]. / Optimación del proceso de producción de la Vacuna Pertussis.

The production of Pertussis Vaccine was reevaluated at the Instituto Nacional de Higiene "Rafael Rangel" in order to optimise it in terms of vaccine yield, potency, specific toxicity and efficiency (cost per doses). Four different processes, using two culture media (Cohen-Wheeler and Fermentación Glutamato Prolina-1) and two types of bioreactors (25 L Fermentador Caracas and a 450 L industrial fermentor) were compared. Runs were started from freeze-dried strains (134 or 509) and continued until the obtention of the maximal yield. It was found that the combination Fermentación Glutamato Prolina-1/industrial fermentor, shortened the process to 40 hours while consistently yielding a vaccine of higher potency (7.91 +/- 2.56 IU/human dose) and lower specific toxicity in a mice bioassay. In addition, the physical aspect of the preparation was rather homogeneous and free of dark aggregates. Most importantly, the biomass yield more than doubled those of the Fermentador Caracas using the two different media and that in the industrial fermentor with the Cohen-Wheeler medium. Therefore, the cost per doses was substantially decreased.