An in silico scientific basis for LL-37 as a therapeutic for Covid-19.

A multi-pronged approach with help in all forms possible is essential to completely overcome the Covid-19 pandemic. There is a requirement to research as many new and different types of approaches as possible to cater to the entire world population, complementing the vaccines with promising results. The need is also because SARS-CoV-2 has several unknown or variable facets which get revealed from time to time. In this work, in silico scientific findings are presented, which are indicative of the potential for the use of the LL-37 human anti-microbial peptide as a therapeutic against SARS-CoV-2. This indication is based on the high structural similarity of LL-37 to the N-terminal helix, with which the virus interacts, of the receptor for SARS-CoV-2, Angiotensin Converting Enzyme 2. Moreover, there is positive prediction of binding of LL-37 to the receptor-binding domain of SARS-CoV-2; this is the first study to have described this interaction. In silico data on the safety of LL-37 are also reported. As Vitamin D is known to upregulate the expression of LL-37, the vitamin is a candidate preventive molecule. This work provides the possible basis for an inverse correlation between Vitamin D levels in the body and the severity of or susceptibility to Covid-19, as widely reported in literature. With the scientific link put forth herein, Vitamin D could be used at an effective, medically prescribed, safe dose as a preventive. The information in this report would be valuable in bolstering the worldwide efforts to eliminate the pandemic as early as possible.

A decoy strategy to activate the immune system.

An approach comprising a novel fusion protein and inactivated virus, as a more efficacious vaccine against invading viruses is presented, using SARS-CoV-2 as a most prominent example. The fusion protein consists of the Hepatitis B surface antigen (HBsAg) conjugated to the N-terminal helix (NTH) of Angiotensin-Converting Enzyme 2 (ACE2), which is the receptor for SARS-CoV-2. For vaccination, this fusion protein is to be administered together with the whole killed virus. The NTH would bind to the Receptor Binding Domain (RBD) of the Spike protein of the killed virus. Due to HBsAg acting as a decoy, immune responses would be mounted. Neutralizing antibodies (NAbs) pre-existing in people already vaccinated with the recombinant Hepatitis B vaccine, fresh production of NAbs, and NAbs produced by memory B cells would bind to the HBsAg. This would lead to "presentation" of the killed virus to elements of the immune system at close range. Also, there would be enhanced opsonization and effective antigen presentation. This two-component vaccine could be a platform strategy, wherein HBsAg could be linked to the part of the cellular receptor that any new intractable virus binds to, and is administered together with whole inactivated virus. Now, the same fusion protein, administered by itself to persons with infection, would have therapeutic action, yet by harnessing elements of the immune system. NAbs would bind to the fusion protein as above, the NTH of which would bind to the RBDs of the infecting virus, which, in effect would be neutralized.

Immunologic properties of human dermal fibroblasts.

Human dermal fibroblasts are known not to express human leukocyte antigen (HLA)-DR message or protein in the absence of interferon (IFN)-γ. To use allogeneic dermal fibroblasts for cell therapy, as a revalidation, the cells at passage 12 were analyzed for HLA-DR mRNA and surface protein. Although no significant HLA-DR surface protein was found, HLA-DR mRNA expression was observed, without interferon-γ. At an early passage, although HLA-DR surface protein was not found, prominent expression of HLA-DR mRNA was observed without IFN-γ stimulation, which was not typical of dermal fibroblasts studied so far. Intracytoplasmic HLA-DR protein was also not detected, which suggests that the mRNA was not translated. There was no marked stimulation of T-cell proliferation by the fibroblasts in the absence or presence of IFN-γ. Interestingly, indoleamine dioxygenase, a molecule involved in immunosuppression, was also expressed in dermal fibroblasts in the absence of IFN-γ.

A novel dermal tissue construct: development and in vitro characterization.

A dermal tissue construct composed of human dermal fibroblasts and a chitosan sponge has been developed, targeted towards the treatment of diabetic nonhealing ulcers. The construct has been designed in a way that the dermal fibroblasts are arranged as a three-dimensional sheet adhered entirely on one side of the chitosan sponge. This design would allow maximal diffusion of growth factors from the cells to the wound bed when the construct is applied on the wound with the cellular sheet side making contact with the wound bed. The diffusion of secreted growth factors would take place directly from cells to the wound bed without being impeded by a matrix. The cells are present at a high density in the dermal construct, which would aid in accelerated wound healing. The construct has a porous chitosan sponge base, which would allow gas exchange, and renders the dermal construct very flexible so that it would take the shape of the wound contours well, while having mechanical integrity. The viability of cells in the construct is greater than 90%. The dermal construct produces a high amount of vascular endothelial growth factor, from 42 ng to 31 ng in 24 h. The construct also produces high amounts of Interleukin-8 (IL-8), from 375 ng to 1065 ng in the first 24 h. Both VEGF and IL-8 have important roles in the healing of chronic diabetic ulcers.

Three-dimensional organization of dermal fibroblasts by macromass culture.

The three-dimensional organization of cells by high-cell-seeding-density culture, termed 'macromass culture', is described. By macromass culture, dermal fibroblasts can be made to organize themselves into a unified three-dimensional form without the aid of a scaffold, and macroscopic constructs, named macromasses, can be made wholly from cells. The sole factor causing three-dimensional organization is culture of cells at high cell seeding density per unit area. No scaffold or extraneous matrix is used for the generation of macromasses; they are of completely cellular origin. No other agents or external influences such as tissue-inducing chemicals, tissue-inducing growth factors, substratum with special properties, rotational culture, centrifugation etc. are employed for macromass formation, and all seeded cells become part of the cohesive construct. These three-dimensional constructs have the potential for use as in vitro tissue analogues, and a possible application for in vitro cytotoxicity testing is demonstrated.

A novel CD28 mRNA variant and simultaneous presence of various CD28 mRNA isoforms in human T lymphocytes.

The primary transcript of the human CD28 gene in T lymphocytes, encoding for a costimulatory molecule, is known to undergo alternative splicing, and different small sets of variant isoforms have been reported. This report presents the novel simultaneous presence of eight different mRNA isoforms, all observed together in normal human T cells; this is an interesting finding in the study of CD28 mRNA structural variants. A similar pattern was found in a total of four individuals. In addition, we also report the occurence and sequence of a new CD28 mRNA isoform, one of the above eight, which is a novel variant generated by the use of a new combination of splice donor and acceptor sites.