Evaluation of Direct and Cell-Mediated Lactoferrin Gene Therapy for the Maxillofacial Area Abscesses in Rats

Abstract

Resistance to antibacterial therapy requires the discovery of new methods for the treatment of infectious diseases. Lactoferrin (LTF) is a well-known naïve first-line defense protein. In the present study, we suggested the use of an adenoviral vector (Ad5) carrying the human gene encoding LTF for direct and cell-mediated gene therapy of maxillofacial area phlegmon in rats. Abscesses were developed by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. At 3-4 days after phlegmon maturation, all rats received ceftriaxone and afterward were subcutaneously injected around the phlegmon with: (1) Ad5 carrying reporter gfp gene encoding green fluorescent protein (Ad5-GFP control group), (2) Ad5 carrying LTF gene (Ad5-LTF group), (3) human umbilical cord blood mononuclear cells (UCBC) transduced with Ad5-GFP (UCBC + Ad5-GFP group), and (4) UCBC transduced with Ad5-LTF (UCBC + Ad5-LTF group). Control rats developed symptoms considered to be related to systemic inflammation and were euthanized at 4-5 days from the beginning of the treatment. Rats from therapeutic groups demonstrated wound healing and recovery from the fifth to seventh day based on the type of therapy. Histological investigation of cervical lymph nodes revealed purulent lymphadenitis in control rats and activated lymphatic tissue in rats from the UCBC + Ad5-LTF group. Our results propose that both approaches of LTF gene delivery are efficient for maxillofacial area phlegmon recovery in rats. However, earlier wound healing and better outcomes in cervical lymph node remodeling in the UCBC + Ad5-LTF group, as well as the lack of direct exposure of the viral vector to the organism, which may cause toxic and immunogenic effects, suggest the benefit of cell-mediated gene therapy.

Keywords: adenoviral vector; gene therapy; human umbilical cord blood mononuclear cell; lactoferrin gene; lymph node; maxillofacial area phlegmon; rat; wound healing.

Figure 1

Experimental design. A—Acute peritonitis modeling. B—Phlegmon modeling by injection of the purulent peritoneal exudate in the molar region of the medial surface of the mandible. C—Introduction of one of the preparations after the maturation of the abscess.

Figure 2

Expression of transgenes in UCBC 72 h after transduction with Ad5-LTF or Ad5-GFP (multiplicity of infection (MOI) = 10). (A)—fluorescence microscopy shows a specific green signal in UCBC + Ad5-GFP. Cell nuclei were stained with Hoechst 33342. (B)—quantitative analysis of LTF and reporter gfp genes expression level in UCBC + Ad5-LTF and UCBC + Ad5-GFP in comparison with naïve UCBC. Data are presented as mean ± standard error, * p < 0.05.

Figure 3

Phlegmon regression. (A)—Rat from the control group (Ad5-GFP) on the fourth day of treatment. (B)—Rats seven days after injection of UCBC + Ad5-GFP. (C,D)—Rats from the Ad5-LTF and UCBC + Ad5-LTF groups on the sixth day of treatment.

Figure 4

Hematoxylin and eosin staining of rat cervical lymph nodes. Purulent lymphadenitis (A) with a focus of destruction of lymphoid tissue (A’) in rats from the control Ad5-GFP group. Primary lymphoid follicle without a reactive center (B) and monocytoid cells (free macrophages) and lymphocytes in the sinuses (B’) in rats from the UCBC + Ad5-GFP group. An attenuated lymphoid follicle (C) and sinus lymphocytosis: a large number of lymphocytes in the dilated intermediate sinuses (C’) in rats from the Ad5-LTF group. Hyperplastic lymphoid follicle with a large germinal center (D) and sinus histiocytosis (D’) in rats from the UCBC + Ad5-LTF group.

Figure 5

Results of morphometric analysis of rat cervical lymph nodes. Bar plots with error bars illustrate destruction (necrotic mass), preservation (primary follicles and paracortical zone) and activation (secondary follicles) of lymph nodes from the control (Ad5-GFP) and therapeutic (Ad5-LTF, UCBC + Ad5-GFP, and UCBC + Ad5-LTF) groups. Data are presented as mean ± standard error. The dotted lines correspond to the average relative area in intact rats. * p < 0.05 in comparison with the Ad5-GFP group.

Figure 6

Homing UCBC and expression of reporter GFP in rat cervical lymph nodes. Immunofluorescent staining of lymph nodes with an antibody (Ab) to the human nuclear antigen (HNA) in UCBC + Ad5-GFP group (A) and in UCBC + Ad5-LTF group (B). Arrows indicate HNA-positive cells (red) 7 days after UCBC injection, a green signal corresponds GFP. (C)—Fluorescent microscopy of a lymph node from the Ad5-GFP group. Arrows indicate specific fluorescent signal of GFP in host lymphatic cells five days after injection of Ad5 carrying reporter gene, encoding GFP. Cell nuclei were counterstained with DAPI (blue) Scale bar in (A–C) = 50 μm; scale bar in the insert = 20 μm.