VP22 herpes simplex virus protein can transduce proteins into stem cells

The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.

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.

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.

Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

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.

Evaluation of some residual bioactivities of microencapsulated Phaseolus lunatus protein fraction with carboxymethylated flamboyant (Delonix regia) gum/sodium alginate

Recent studies have shown the beneficial effect of peptides, an unexploited source could be Phaseolus lunatus being an important raw material for those functional products in order to improve their utilization. In addition to improve the beneficial effect of bioactive peptides the microencapsulation could be a way to protect the peptides against the environment to which they are exposed. P. lunatus protein fraction (<10 kDa of weight) was encapsulated using a blend of carboxymethylated flamboyant gum (CFG) and sodium alginate (SA) at different concentrations of CaCl2 and hardening times. After in vitro digestion of microcapsules the residual activity, in the intestinal system, both inhibition of agiotensin-converting enzyme (I-ACE) and antioxidant activity obtained were in a range of 0.019-0.136 mg/mL and 570.64-813.54 mM of TEAC respectively. The microencapsulation employed CFG/SA blends could be used controlled delivery of peptide fractions with potential use as a nutraceutical or therapeutic agents.

Nutritional factors in milk from Brazilian mothers delivering small for gestational age neonates

The composition of breast milk from brazilian mothers delivering low birthweight infants and its adequacy as a source of nutrients for this group has not yet been fully elucidated. A total of 209 milk samples from 66 women were analysed. The mothers were divided into three groups: G1, mothers delivering term babies of low birthweight (TSGA, n=16); G2, mothers delivering preterm babies of appropriate birthweight (PTAGA, n=20); G3, mothers delivering term babies of appropriate birthweight (TAGA, n=30). The following factors were analysed: osmolarity, total proteins and protein fractions, creamatocrit, sodium, potassium, calcium and magnesium. Milk samples were collected 48 h and 7, 15, 30 and 60 days after delivery. The groups did not differ significantly in terms of osmolarity, total proteins and fractions, creamatocrit, calcium, magnesium or potassium throughout the study period. Sodium levels were higher in all samples from mothers of TSGA infants and in samples from mothers of PTAGA infants on the 7th, 15th and 30th days than in milk from the TAGA group. The authors consider the needs of the low birthweight and TAGA infants and that these high sodium levels may be necessary for growth of low birthweight infants.

Risk factors for the increasing trend in low birth weight among live births born by vaginal delivery, Brazil

OBJECTIVE: To identify risk factors for low birth weight (LBW) among live births by vaginal delivery and to determine if the disappearance of the association between LBW and socioeconomic factors was due to confounding by cesarean section. METHODS: Data were obtained from two population-based cohorts of singleton live births in Ribeirão Preto, Southeastern Brazil. The first one comprised 4,698 newborns from June 1978 to May 1979 and the second included 1,399 infants born from May to August 1994. The risks for LBW were tested in a logistic model, including the interaction of the year of survey and all independent variables under analysis. RESULTS: The incidence of LBW among vaginal deliveries increased from 7.8% in 1978--79 to 10% in 1994. The risk was higher for: female or preterm infants; newborns of non-cohabiting mothers; newborns whose mothers had fewer prenatal visits or few years of education; first-born infants; and those who had smoking mothers. The interaction of the year of survey with gestational age indicated that the risk of LBW among preterm infants fell from 17.75 to 8.71 in 15 years. The mean birth weight decreased more significantly among newborns from qualified families, who also had the highest increase in preterm birth and non-cohabitation. CONCLUSIONS: LBW among vaginal deliveries increased mainly due to a rise in the proportion of preterm births and non-cohabiting mothers. The association between cesarean section and LBW tended to cover up socioeconomic differences in the likelihood of LBW. When vaginal deliveries were analyzed independently, these socioeconomic differences come up again.

Trends in the modes of delivery and their impact on perinatal mortality rates

OBJECTIVE: To determine changes in the incidence of vaginal deliveries, cesarean sections, and forceps deliveries and their potential association with fetal, early neonatal, and perinatal mortality rates over time. METHODS: A retrospective study was carried out and the occurrence of deliveries supervised by university services between January 1991 and December 2000 was determined. Data regarding fetal, early neonatal, and perinatal deaths were assessed using obstetric and pediatric records and autopsy reports. RESULTS: Of a total of 33,360 deliveries, the incidence of vaginal deliveries, cesarean sections, and forceps deliveries was relatively steady (around 60, 30, and 10%, respectively) while, at the same time, there was a marked reduction in fetal mortality (from 33.3 to 13.0‰), early neonatal mortality (from 30.6 to 9.0‰), and perinatal mortality (from 56.4 to 19.3‰). CONCLUSIONS: The marked reduction in perinatal mortality rates seen during the study period without an increase in cesarean sections indicates that the decrease in perinatal mortality was not impacted by cesarean section rates. The plausible hypothesis seems to be that the reduction in perinatal mortality of deliveries performed under the supervision of university services was more likely to be associated with better neonatal care rather than the mode of delivery.

Obesity and gestational weight gain: cesarean delivery and labor complications

OBJECTIVE: To assess the association between pre-gestational obesity and weight gain with cesarean delivery and labor complications. METHODS: A total of 4,486 women 20-28 weeks pregnant attending general prenatal care clinics of the national health system in Brazil from 1991 to 1995 were enrolled and followed up through birth. Body mass index categories based on prepregnancy weight and total weight gain were calculated. Associations between body mass index categories and labor complications were adjusted through logistic regression analysis. RESULTS: Obesity was present in 308 (6.9%) patients. Cesarean delivery was performed in 164 (53.2%) obese, 407 (43.1%) pre-obese, 1,045 (35.1%) normal weight and 64 (24.5%) underweight women. The relative risk for cesarean delivery in obese women was 1.8 (95% CI: 1.5-2.0) compared to normal weight women. Greater weight gain was particularly associated with cesarean among the obese (RR 4th vs 2nd weight gain quartile 2.2; 95% CI: 1.4-3.2). Increased weight at the beginning of pregnancy was associated with a significantly higher adjusted risk of meconium with vaginal delivery and perinatal death and infection in women submitted to cesarean section. Similarly, greater weight gain during pregnancy increased the risk for meconium and hemorrhage in women submitted to vaginal delivery and for prematurity with cesarean. CONCLUSIONS: Pre-gestational obesity and greater weight gain independently increase the risk of cesarean delivery, as well as of several adverse outcomes with vaginal delivery. These findings provide further evidence of the negative effects of prepregnancy obesity and greater gestational weight gain on pregnancy outcomes.

Herpes simplex virus type 2 infection in pregnancy: asymptomatic viral excretion at delivery and seroepidemiologic survey of two socioeconomically distinct populations in São Paulo, Brazil

The objective of the present study was to estimate the prevalence of herpes simplex virus type 2 (HSV 2) antibodies in child bearing women of 2 Brazilian populations with different socioeconomic status and to determine the risk of neonatal HSV exposure by means of maternal cultures at the onset of labor. The study was conducted at 2 hospitals: A, serving very low income patients and B, serving middle socioeconomic class. 173 participants from group A and 127 from B answered a questionnaire which showed that the patients had similar ages (27.7 and 26.8 years, respectively) but differed with regard to socioeconomic status, age at first intercourse (18.6 vs 20.6 years), number of sex partners (1.5 vs 1.2) and previous sexually transmitted diseases (15% vs. 1.5%). History of genital herpes was given by 11% of group A participants and by a similar number, 7%, of patients from group B. In addition, 200 serum samples from population A and 455 from B were tested by ELISA for and HSV antibodies and 92% and 86%, respectively, were found to be positive. Sixty seropositive samples from group A and 90 from B were further analyzed by Western blot, which showed the presence of type 2 specific antibodies in 46% and 36%, respectively, suggesting an overall HSV 2 prevalence of 42% in group A and 31% in B. Cervical specimens were obtained for culture from 299 asymptomatic patients of population A and 313 of B. HSV was isolated from one specimen in each group, indicating a 0.3% incidence of asymptomatic viral excretion in both populations. In conclusion, the prevalence of type 2 antibodies in childbearing women was very high, but it did not differ with the socioeconomic status. The risk of HSV perinatal transmission was also similar in the 2 study populations and it was comparable with the data from developed countries. Our findings do not indicate the need of special screening programs for asymptomatic HSV excretion in Brazilian pregnant women.