Structure-activity relationship study on a simple cationic peptide motif for cellular delivery of antisense peptide nucleic acid

Improving cellular uptake and biodistribution remains one of the major obstacles for a successful and broad application of peptide nucleic acids (PNAs) as antisense therapeutics. Recently, we reported the identification and functional characterization of an antisense PNA, which redirects splicing of murine CD40 pre-mRNA. In this context, it was discovered that a simple octa(l-lysine) peptide covalently linked to the PNA is capable of promoting free uptake of the conjugate into BCL1 cells as well as primary murine macrophages. On the basis of this peptide motif, the present study aimed at identifying the structural features, which define effective peptide carriers for cellular delivery of PNA. While the structure-activity relationship study revealed some clear correlations, only a few modifications actually led to an overall improvement as compared to the parent octa(l-lysine) conjugate. In a preliminary PK/tissue distribution study in healthy mice, the parent conjugate exhibited relatively broad tissue distribution and only modest elimination via excretion within the time frame of the study.     

Missed opportunities for prevention of mother-to-child transmission of human immunodeficiency virus type 1 in Latin America and the Caribbean: the NISDI perinatal study

Cases of mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) in a prospective cohort study in Latin America and the Caribbean were analyzed. Eight of 820 eligible infants became infected [transmission rate, 0.98% (95% CI = 0.45-1.96%)]. Five cases (62%) represented missed opportunities for prevention of MTCT of HIV-1, suggesting the need for ongoing training and education of clinicians regarding prevention of MTCT of HIV-1.     

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.Monoclonal antibodies (mAbs) are a rapidly growing class of therapeutics that combine high binding affinities and specificities with long in vivo half-lives. A large number of mAbs have been approved for therapeutic use or are in development (1–6). Early therapeutic mAbs were derived from mouse sources, retained mouse sequences, and were thus immunogenic when used in human patients, limiting the ability to dose repeatedly. The use of humanized and/or fully human antibodies avoided immunogenicity problems and allowed long-term treatment of chronic diseases. Thus, a variety of systems were developed to create humanized or fully human therapeutic antibodies (7–18). One popular approach involves the in vitro isolation of antigen-specific antibodies using display strategies involving human antibody fragments expressed on the surface of phage, bacteria, or yeast (12). Although these synthetic approaches can be quite powerful and can rapidly generate leads, they potentially result in increased immunogenicity in vivo, and the initial display-derived antibody fragments can subsequently require extensive post hoc protein reengineering efforts when reformatted into conventional antibody formats to overcome issues such as insolubility, aggregation, and proteolysis (1, 12, 19). Natural selection of antibodies in vivo within mammalian systems tends to optimize desirable biochemical and pharmacokinetic properties, avoiding the need for extensive post hoc reengineering. Thus, as first proposed based on the finding that human Ig genes efficiently rearrange when introduced into mouse pre-B cells (20), another popular approach for generating human therapeutic mAbs was developed using transgenic mice genetically engineered to produce fully human antibodies (15–18). These so-called HumAb mice were engineered using a “KO-plus-transgenic” strategy in which the endogenous murine Ig genes were disrupted to eliminate the endogenous mouse immune response, whereas transgenic introduction of human Ig loci at different random genetic loci drove production of fully human antibodies.Although HumAb mice generated using this KO-plus-transgenic approach represented a transforming advance in the field, they suffered, however, from partial immune deficiencies compared with WT mice, limiting their ability to produce robust Ab responses to some antigens (21–23). The immune deficiencies of these first-generation HumAb mice may be due to the manner in which they were genetically engineered. First, the genomic context of the randomly inserted human transgenes may contribute to their inefficient functionality, as they may lack extended locus control regions such as the 3′ enhancers (24) and regulatory region (25) of the Ig heavy (IgH) locus, which have been shown to play critical roles in Ig expression and class switching or even alter the 3D location of the Ig genes within B-cell nuclei (26, 27) and thus perturb function in unanticipated ways. In addition, the immune deficiencies of the first-generation HumAb mice may be partly explained by their use of human constant regions. Immunoglobulins interact with other components of the B-cell receptor (BCR) signaling complex via their constant regions, and such interactions are required for appropriate signaling required for antigen-independent B-cell development in bone marrow, as well as antigen-dependent natural selection processes in the periphery (28–36). Others previously noted that interactions between constant regions and BCR coreceptors do not operate efficiently across species (29). Thus, we reasoned that the immune deficiencies in first-generation HumAb mice may in part be due to inefficient interspecies protein-protein interactions between human constant regions and the mouse coreceptors. Finally, the secreted human immunoglobulins produced in the HumAb mice may also interact inefficiently with various mouse Fc receptors, further adversely affecting the humoral immune response (37, 38).As described in the companion article (39), we attempted to exploit the advantages and overcome the limitations of the first-generation HumAb approaches by precisely replacing the entire mouse germ-line variable region gene repertoire with the equivalent human germ-line variable sequences in situ, while maintaining all mouse constant regions and all known gene expression control elements within the natural mouse genomic location. We reasoned that the introduced human Ig variable gene segments would rearrange normally, be linked to mouse constant regions, and furthermore be expressed from the endogenous mouse Ig loci at physiologically appropriate levels. Because these “reverse chimeric” antibody molecules would bear human antigen-binding variable domains fused to mouse constant domains, we presumed they would interact in a species appropriate way with mouse BCR coreceptors and mouse Fc receptors, resulting in a fully functional immune system. We refer to these humanized Ig variable domain mice as VelocImmune mice because they were generated using VelociGene technology (40).In this paper, we show that VelocImmune mice have a humoral immune system indistinguishable from that of WT mice, with normal cell populations at all stages of B-cell development and normal lymphoid organ structures. Sequences of antibodies derived from the humanized Ig loci exhibit normal variable segment rearrangement, somatic hypermutation, and class switching. Immunizations of VelocImmune mice generate robust humoral responses from which a large diversity of monoclonal antibodies can be isolated. Thus, VelocImmune mice are a unique platform for the efficient production of fully human antibodies, several of which have already entered clinical development.     

Group B Streptococcus in a cohort of HIV-infected pregnant women: prevalence of colonization, identification and antimicrobial susceptibility profile

Group B Streptococcus (GBS) is a leading cause of infectious morbidity in newborns. We describe the prevalence of GBS colonization and the serotypes and antibiotic susceptibility profiles of isolates obtained from a cohort of human immunodeficiency virus (HIV)-infected pregnant women. This was a cross-sectional study at a centre for the prevention of mother-to-child transmission of HIV. Vaginal and rectal swabs were collected at 35-37 weeks of gestation from 158 eligible women. GBS isolates were serotyped and antimicrobial susceptibility tests performed. Patient sociodemographic characteristics, CD4 counts and viral loads were abstracted from records. The overall anogenital prevalence of GBS colonization was 49/158 (31.0%): 40/158 (25.3%) for vagina, 19/158 (12.0%) for rectum and 10/158 (6.3%) for both. Predominant serotypes were Ib (34.9%) and Ia (25.6%). All were penicillin-susceptible. Two were resistant to erythromycin (4.0%) and one to clindamycin (2.0%). The colonization rate by GBS was high in this cohort. Serotype Ib was the most frequently identified.     

Accuracy of a rapid real-time polymerase chain reaction assay for diagnosis of group B Streptococcus colonization in a cohort of HIV-infected pregnant women

Objective: There are limited data regarding Xpert performance to detect Group B Streptococcus (GBS) in HIV-infected pregnant women. We evaluated the accuracy of a rapid real-time polymerase chain reaction (PCR) test in a cohort of HIV-infected women.

Methods: At 35–37 weeks of pregnancy, a pair of combined rectovaginal swabs were collected for two GBS assays in a cohort of sequentially included HIV-infected women in Rio de Janeiro: (1) culture; and (2) real-time PCR assay [GeneXpert GBS (Cepheid, Sunnyvale, CA)]. Using culture as the reference, sensitivity, specificity, positive and negative-likelihood ratios were estimated.

Results: From June 2012 to February 2015, 337 pregnant women met inclusion criteria. One woman was later excluded, due to failure to obtain a result in the index test; 336 were included in the analyses. The GBS colonization rate was 19.04%. Sensitivity and specificity of the GeneXpert GBS assay were 85.94% (95% CI: 75.38–92.42) and 94.85% (95% CI: 91.55–96.91), respectively. Positive and negative predictive values were 79.71% (95% CI: 68.78–87.51) and 96.63% (95% CI: 93.72–98.22), respectively.

Conclusions: GeneXpert GBS is an acceptable test for the identification of GBS colonization in HIV-infected pregnant women and represents a reasonable option to detect GBS colonization in settings where culture is not feasible.     

¿Es necesaria la orquidectomía en los pacientes con testículo no descendido post-puberal?

ObjectiveThe cryptorchidism is a risk factor for testicular cancer and is associated with cancer in the contraleteral testicle. The mechanism that leads to an increased risk of causes in a cryptorchid testis is unclear.Materials and MethodsWe analyzed 25 patients of 20-years old for maldescended testicles we practiced orchiectomy in 22 testicles and orchiopexy in 7 testicles. The time patients had the testicles in an anomalous location were between 20-51 years.ResultsThe elder patients with maldescends testicles came because the couldn’t feel the testicle in the scrotum and the 16% of them because of infertility. Only 1 of 25 cases (4%) were demonstrated cancer with pure seminoma.ConclusionsIn the following up of the patients none of them presented contralateral testicle cancer between 5-10 years. The patients who were practiced the orchiopexy no-one presented testicular cancer between 5-11 years.     

Family Centered Intrapartum Care: Revisiting an Old Concept

The historic development of family centered care to the present time is traced, and future development is proposed. A family systems approach can help nurses focus on the family as a unit and on individual members. This requires nurses to develop skills in working with multiple family members, provide care and advocacy for varying and sometimes conflicting family needs, and promote family clarification of their own values and priorities. Such an approach can help intrapartum families add to their experience of coping with stress in a way that builds family cohesion and mutual support.