Nasal delivery of an IgM offers broad protection from SARS-CoV-2 variants.

Resistance represents a major challenge for antibody-based therapy for COVID-191-4. Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.

Role of protein kinase C alpha in calcium induced keratinocyte differentiation: defective regulation in squamous cell carcinoma.

Calcium induces both involucrin and transglutaminase-K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31-8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium-and time-dependent manner. PKC [epsilon] decreased in a time-and calcium-dependent fashion over the three-week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium-regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium-induced differentiation, PKC [alpha] plays a necessary role in mediating calcium-induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells.

Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth.

The active vitamin D metabolite, 1,25-dihydroxyvitamin D, acting through the vitamin D receptor, regulates the expression of genes in a variety of vitamin D-responsive tissues, including the epidermis. To investigate the role of the vitamin D receptor in mediating epidermal differentiation, we examined the histomorphology and expression of differentiation markers in the epidermis of vitamin D receptor knockout mice generated by gene targeting. The homozygous knockout mouse displayed a phenotype that closely resembles vitamin D-dependent rickets type II in humans, including the development of rickets and alopecia. Hair loss developed by 3 mo after birth and gradually led to nearly total hair loss by 8 mo. Histologic analysis of the skin of homozygous knockout mice revealed dilation of the hair follicles with the formation of dermal cysts starting at the age of 3 wk. These cysts increased in size and number with age. Epidermal differentiation markers, including involucrin, profilaggrin, and loricrin, detected by immunostaining and in situ hybridization, showed decreased expression levels in homozygous knockout mice from birth until 3 wk, preceding the morphologic changes observed in the hair follicles. Keratin 10 levels, however, were not reduced. At the ultrastructural level, homozygous knockout mice showed increased numbers of small dense granules in the granular layer with few or no surrounding keratin bundles and a loss of keratohyalin granules. Thus, both the interfollicular epidermis and the hair follicle appear to require the vitamin D receptor for normal differentiation. The temporal abnormalities between the two processes reflect the apparent lack of requirement for the vitamin D receptor during the anagen phase of the first (developmental) hair cycle, but with earlier effects on the terminal differentiation of the interfollicular epidermis.