Ophthalmic alterations in horses with leptospirosis by serovar Icterohaemorrhagiae in Rio de Janeiro, Brazil

The objective of the study was to determine the association between clinical ophthalmic alterations and seroreactivity to leptospirosis by serovar Icterohaemorrhagiae in horses in Rio de Janeiro, Brazil. A total of 199 horses were studied. A microscopic agglutination test (MAT) was used to detect specific anti-Leptospira antibodies in blood serum. A total of 107 (53.8%) horses were seroreactive (titres > 200); 54 had high (> 800) titres, of which 44 were against serovar Icterohaemorrhagiae. Forty-two out of these 44, plus 40 seronegative horses (titers < 100) were given detailed ophthalmic examinations. Epiphora, ocular congestion, blepharospasm, photophobia, and peripapillary focal depigmentation were the most frequent alterations in seroreactive horses. Most ocular alterations were significantly more frequent in seroreactive horses. Horses seroreactive for leptospirosis (serovar Icterohaemorrhagiae) had a significantly higher prevalence of ophthalmic alterations than seronegative horses, providing additional evidence for an association between leptospirosis and equine uveitis.

Factors affecting immunoglobulin concentration in colostrum of healthy Holstein cows immediately after delivery

This study analyzed the influence of the number of milkings, number of births, and udder quarter in immunoglobulin (Ig) concentration in the colostrum of healthy Holstein cows. It was collected two samples of colostrum by manual milking, getting the first jets to completion of bacteriological examination and immunoglobulin levels by radial immunodiffusion test in agar gel. Positive samples for bacteriological examination were excluded from this investigation. Medians of immunoglobulin's G, A and M in the colostrum collected before the first and second milking were respectively 9,200 and 6,400mg/dL (p=0.0029); 400 and 200mg/dL (p=0.0018); 800 and 400mg/dL (p=0.0001). Median immunoglobulin concentration in animals that calved once, twice or three times or in cows that calved 4 to 6 times were 6,400; 6,400; 3,200 and 11,200mg/dL IgG; 100, 200, 100 and 800mg/dL IgA ; and 400, 400, 100 and 800mg/dL IgM, respectively. Concentrations of IgG, IgA and IgM were greater in animals that calved more than 4 times (p<0.05). Medians of IgG, IgA and IgM in the right fore quarter (RF), right hind quarter (RH), left fore quarter (LF) and left hind quarter (LH) were, respectively, 7,800; 6,400; 7,800 and 6,400mg/dL; 200, 200, 200 and 200mg/dL; and 400, 400, 400 and 400mg/dL. Ig concentrations in the colostrum of Holstein cows were influenced by the number of milkings after delivery and number of lactations. These variations may be considered risk factors to passive immunity transfer to newborn calves, predisposing them to diseases and causing economic losses to dairy production.

Liposomes as a gene delivery system

Gene therapy is an active field that has progressed rapidly into clinical trials in a relatively short time. The key to success for any gene therapy strategy is to design a vector able to serve as a safe and efficient gene delivery vehicle. This has encouraged the development of nonviral DNA-mediated gene transfer techniques such as liposomes. Many liposome-based DNA delivery systems have been described, including molecular components for targeting given cell surface receptors or for escaping from the lysosomal compartment. Another recent technology using cationic lipids has been evaluated and has generated substantial interest in this approach to gene transfer.

Poly-DL-lactide-co-glycolide microspheres as a controlled release antigen delivery system

Successful vaccine application means maximum protection with minimal number of administrations. A rational development of vaccines involves studies of the nature of the antigen as well as of the adjuvant to be used to improve the immune responses. This has provided the impetus for studies to design the degradable devices and for different approaches to antigen delivery by different routes of administration. The development of controlled release systems based on polymeric devices that permit a sustained or pulsed release of encapsulated antigens has attracted much interest. Polymeric delivery systems consist of polymers that release their content continuously in a controlled manner over a period of time. The development of a biocompatible delivery system for parenteral administration offers several advantages in terms of immunoadjuvanticity over other compounds. It was found that, in contrast to other carriers, microspheres are more stable, thus permitting administration by the oral or parenteral route. In the present study, we describe the main characteristics and potentialities of this new immunoadjuvant for oral and parenteral administration.

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitope-protein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4% and decreased its ß-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Antiretroviral agents and acid-base balance at delivery of the neonate

Limited evidence is available regarding antiretroviral (ARV) safety for uninfected infants exposed to these drugs in utero. Our objective was to determine if ARV administered to pregnant women is associated with decreasing umbilical arterial pH and base excess in uninfected infants. A prospective study was conducted on 57 neonates divided into three groups: ZDV group, born to mothers taking zidovudine (N = 20), triple therapy (TT) group, born to mothers taking zidovudine + lamivudine + nelfinavir (N = 25), and control group (N = 12), born to uninfected mothers. Umbilical cord blood was used to determine umbilical artery gases. A test was performed to calculate the sample by comparing means by the unpaired one-tailed t-test, with a = 0.05 and ß = 20%, indicating the need for a sample of 18 newborn infants for the study groups to detect differences higher than 20%. The control and ARV groups were similar in gestational age, birth weight, and Apgar scores. Values of pH, pCO2, bicarbonate, and base excess in cord arterial blood obtained at delivery from the newborns exposed to TT were 7.23, 43.2 mmHg, 19.5 mEq/L, and -8.5 nmol/L, respectively, with no significant difference compared to the control and ZDV groups. We conclude that intrauterine exposure to ARV is not associated with a pathological decrease in umbilical arterial pH or base excess. While our data are reassuring, follow-up is still limited and needs to be continued into adulthood because of the possible potential for adverse effects of triple antiretroviral agents.

Retinal protective effects of topically administered agmatine on ischemic ocular injury caused by transient occlusion of the ophthalmic artery

Agmatine, an endogenous polyamine and putative neuromodulator, is known to have neuroprotective effects on various neurons in the central nervous system. We determined whether or not topically administered agmatine could reduce ischemic retinal injury. Transient ocular ischemia was achieved by intraluminal occlusion of the middle cerebral artery of ddY mice (30-35 g) for 2 h, which is known to also induce occlusion of the ophthalmic artery. In the agmatine group (N = 6), a 1.0 mM agmatine-containing ophthalmic solution was administered four times daily for 2 weeks before occlusion. In the control group (N = 6), a 0.1% hyaluronic acid ophthalmic solution was instilled at the same times. At 22 h after reperfusion, the eyeballs were enucleated and the retinal sections were stained by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Transient ocular ischemia induced apoptosis of retinal cells in the entire retinal layer, and topically administered agmatine can significantly reduce this ischemic retinal injury. The proportion of apoptotic cells was definitely decreased (P < 0.001; Kruskal-Wallis test). Overall, we determined that topical agmatine application effectively decreases retinal damage in an in vivo ocular ischemic injury model. This implies that agmatine is a good candidate as a direct neuroprotective agent for eyes with ocular ischemic diseases.

Nasal CPAP in the delivery room for newborns with extremely low birth weight in a hospital in a developing country

The objective of this study was to determine the feasibility of the use of continuous positive airway pressure installed prophylactically in the delivery room (DR-CPAP), for infants with a birth weight between 500 and 1000 g in settings with limited resources. During 23 months, infants with a birth weight between 500 and 1000 g consecutively received DR-CPAP. A total of 33 infants with low birth weight were enrolled, 16 (48.5%) were females. Only 14 (42.4%) received antenatal corticosteroids and only 2 of those 14 (14.3%) infants weighing 500-750 g were not intubated in the delivery room, and apnea was given as the reason for intubation of these patients. Of the 19 infants in the 751-1000 g weight range, 9 (47.4%) were intubated in the delivery room, 6 due to apnea and 3 due to respiratory discomfort. For DR-CPAP to be successful, it is probably necessary for preterm babies to be more prepared at birth to withstand the respiratory effort without the need for intubation. Antenatal corticosteroids and better prenatal monitoring are fundamental for success of DR-CPAP.

Application of continuous positive airway pressure in the delivery room: a multicenter randomized clinical trial

This study evaluated whether the use of continuous positive airway pressure (CPAP) in the delivery room alters the need for mechanical ventilation and surfactant during the first 5 days of life and modifies the incidence of respiratory morbidity and mortality during the hospital stay. The study was a multicenter randomized clinical trial conducted in five public university hospitals in Brazil, from June 2008 to December 2009. Participants were 197 infants with birth weight of 1000-1500 g and without major birth defects. They were treated according to the guidelines of the American Academy of Pediatrics (APP). Infants not intubated or extubated less than 15 min after birth were randomized for two treatments, routine or CPAP, and were followed until hospital discharge. The routine (n=99) and CPAP (n=98) infants studied presented no statistically significant differences regarding birth characteristics, complications during the prenatal period, the need for mechanical ventilation during the first 5 days of life (19.2 vs 23.4%, P=0.50), use of surfactant (18.2 vs 17.3% P=0.92), or respiratory morbidity and mortality until discharge. The CPAP group required a greater number of doses of surfactant (1.5 vs 1.0, P=0.02). When CPAP was applied to the routine group, it was installed within a median time of 30 min. We found that CPAP applied less than 15 min after birth was not able to reduce the need for ventilator support and was associated with a higher number of doses of surfactant when compared to CPAP applied as clinically indicated within a median time of 30 min.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

Development and characterization of orally-disintegrating films for propolis delivery

This study aimed at evaluating the effect of different concentrations of hydrolyzed collagen (HC) on the properties of an orally disintegrating film containing propolis ethanol extract (PEE) as an active component. The films were evaluated in terms of total phenols, mechanical properties, solubility, contact angle, disintegration time, and microstructure. The films were prepared by casting with 2 g of protein mass (gelatin and HC), 30 g of sorbitol/100 g of protein mass, and 100 g of PEE/100 g of protein mass. HC was incorporated at concentrations of 0, 10, 20, and 30 g/100 g of protein mass. It was found that increased concentrations of HC reduced tensile strength and increased elongation; however, all films showed plastic behavior. An increase in solubility at 25 ºC, a reduction in the contact angle, and disintegration time were also observed. Thus, higher concentrations of collagen led to more hydrophilic and more soluble polymeric matrices that showed shorter dissolution time, favoring the use of these materials as carriers for active compounds to be delivered in the oral cavity.