# Roles of D-Amino Acids on the Bioactivity of Host Defense Peptides
## 6 In Vivo Testing of d-AA-Containing HDPs
### Targeting Other Pathogens  
There are only a limited number of in vivo tests that have been carried out for evaluating the activity of d-AA-containing HDPs against diseases other than bacterial infection and cancer. One interesting study, despite being ex vivo rather than in vivo was carried out by [PERSON] et al. [4], which demonstrated the capacity of the d-enantiomer of BMAP-28 (i.e., a cathelicidin analogue) in neutralizing the protozoan _Leishmania_ inside living macrophages. _Leishmania_ possesses two forms, the free living promastigotesote form and the parasitic intracellular amastigotes form. _Leishmania_ is typically treated with polyvalent antimony. However, increased incidence of antimony resistance by _Leishmania_ has been observed [118]. Previous works have demonstrated the effectiveness of various HDPs at neutralizing _Leishmania_ promastigotes via the characteristic HDP membrane depolarization; however, there were no reports of activity against the intracellular amastigotes form. The study by [PERSON] et al. [4] demonstrated that both l- and d-enantiomers of BMAP-28 were able to greatly reduce the _Leishmania_ amastigotes burden inside mouse macrophages in which the d-enantiomer was twice as effective as the l-enantiomer. In vitro tests showed that both the d and l forms of BMAP-28 could neutralize _Leishmania_ promastigotes via membrane depolarization and late-stage apoptosis. However, this study did not explain how the peptide works against _Leishmania_ amastigotes and how the peptide enters the infected macrophage.  
In addition to treating infections in animals, HDPs have been found to defend plants both locally and systemically from bacterial and fungal infection. [PERSON] et al. [119] synthesized cationic diastereomeric peptides consisting of only four residues conjugated to fatty acids. According to the study, this type of lipoprotein is considered a subfamily of HDPs. These lipoproteins can act via membrane lysis and can protect against bacterial and fungal infection when applied directly to the infected part of the plant while not harming the plant tissue [120]. Additionally, the peptides could prime the systemic induction of antimicrobial compounds. In the study by [PERSON] et al. [119], _Arabidopsis_ plant seedlings and cucumber leaves were rendered resistant to _P. syringae_ bacteria and _B. cinerea_ fungi if suspended in the peptide solution 24 h before bacterial challenge. The peptide afforded no protection if both the bacteria and peptide were suspended together with the seedling, indicating that the peptide worked more like a vaccine. A gene expression assay revealed the activation of defense-related genes and the systemic induction of antimicrobial compounds. The defensive effects induced by peptide treatment lasted at least 4 days. [PERSON] et al. [121] synthesized a series of all-d enantiomeric hexappeptides through the screening of a synthetic peptide combinatorial library in a positional scanning format. The peptides were tested in vivo for their capacity to delay the decay of orange fruits via a wound infection assay. One of the peptides could keep the wound free of infection in 38% of the cases for 3 days.