Rob R. Eversole

Filarial Excretory-Secretory Products Induce Human Monocytes to Produce Lymphangiogenic Mediators

The nematodes Wuchereria bancrofti and Brugia spp. infect over 120 million people worldwide, causing lymphedema, elephantiasis and hydrocele, collectively known as lymphatic filariasis. Most infected individuals appear to be asymptomatic, but many exhibit sub-clinical manifestations including the lymphangiectasia that likely contributes to the development of lymphedema and elephantiasis. As adult worm excretory-secretory products (ES) do not directly activate lymphatic endothelial cells (LEC), we investigated the role of monocyte/macrophage-derived soluble factors in the development of filarial lymphatic pathology. We analyzed the production of IL-8, IL-6 and VEGF-A by peripheral blood mononuclear cells (PBMC) from naïve donors following stimulation with filarial ES products. ES-stimulated PBMCs produced significantly more IL-8, IL-6 and VEGF-A compared to cells cultured in medium alone; CD14+ monocytes appear to be the primary producers of IL-8 and VEGF-A, but not IL-6. Furthermore, IL-8, IL-6 and VEGF-A induced in vitro tubule formation in LEC Matrigel cultures. Matrigel plugs supplemented with IL-8, IL-6, VEGF-A, or with supernatants from ES-stimulated PBMCs and implanted in vivo stimulated lymphangiogenesis. Collectively, these data support the hypothesis that monocytes/macrophages exposed to filarial ES products may modulate lymphatic function through the secretion of soluble factors that stimulate the vessel growth associated with the pathogenesis of filarial disease.

The Association of adult onchocerca volvulus with lymphatic vessels

Abstract Immunocytochemical examination of onchocercal nodule tissues containing adult Onchocerca volvulus using immuno-markers for blood and lymphatic vessels (vWF, D2-40, podoplanin, Prox-1, and Lyve1) shows a distinct pattern of distribution of these vessels within nodules. Blood vessels were commonly seen associated with organized lymphoid cellular aggregates in the both the outer and inner areas of the nodules. In contrast, the majority of the lymphatic vessel positivity was seen in the central zone in close apposition to the adult parasites, and the remainder usually associated with microfilariae in the outer areas of the nodule. These findings suggest an intimate relationship between adult O. volvulus and lymphatic vessels, including the likely proliferation of lymphatic endothelial cells (lymphangectasia) akin to that seen with other filariae. These findings indicate that adult O. volvulus may migrate via the lymphatic system, and that clinical manifestations of this disease that involve tissue edema may be the result of the location of these worms in the lymphatic system.

Ivermectin treatment of Loa loa hyper-microfilaraemic baboons (Papio anubis): Assessment of microfilarial load reduction, haematological and biochemical parameters and histopathological changes following treatment

Background Individuals with high intensity of Loa loa are at risk of developing serious adverse events (SAEs) post treatment with ivermectin. These SAEs have remained unclear and a programmatic impediment to the advancement of community directed treatment with ivermectin. The pathogenesis of these SAEs following ivermectin has never been investigated experimentally. The Loa/baboon (Papio anubis) model can be used to investigate the pathogenesis of Loa-associated encephalopathy following ivermectin treatment in humans. Methods 12 baboons with microfilarial loads > 8,000mf/mL of blood were randomised into four groups: Group 1 (control group receiving no drug), Group 2 receiving ivermectin (IVM) alone, Group 3 receiving ivermectin plus aspirin (IVM + ASA), and Group 4 receiving ivermectin plus prednisone (IVM + PSE). Blood samples collected before treatment and at Day 5, 7 or 10 post treatment, were analysed for parasitological, hematological and biochemical parameters using standard techniques. Clinical monitoring of animals for side effects took place every 6 hours post treatment until autopsy. At autopsy free fluids and a large number of standard organs were collected, examined and tissues fixed in 10% buffered formalin and processed for standard haematoxylin-eosin staining and specific immunocytochemical staining. Results Mf counts dropped significantly (p<0.05) in all animals following ivermectin treatment with reductions as high as (89.9%) recorded; while no significant drop was observed in the control animals. Apart from haemoglobin (Hb) levels which recorded a significant (p = 0.028) drop post treatment, all other haematological and biochemical parameters did not show any significant changes (p>0.05). All animals became withdrawn 48 hours after IVM administration. All treated animals recorded clinical manifestations including rashes, itching, diarrhoea, conjunctival haemorrhages, lymph node enlargement, pinkish ears, swollen face and restlessness; one animal died 5 hours after IVM administration. Macroscopic changes in post-mortem tissues observed comprised haemorrhages in the brain, lungs, heart, which seen in all groups given ivermectin but not in the untreated animals. Microscopically, the major cellular changes seen, which were present in all the ivermectin treated animals included microfilariae in varying degrees of degeneration in small vessels. These were frequently associated with fibrin deposition, endothelial changes including damage to the integrity of the blood vessel and the presence of extravascular erythrocytes (haemorrhages). There was an increased presence of eosinophils and other chronic inflammatory types in certain tissues and organs, often in large numbers and associated with microfilarial destruction. Highly vascularized organs like the brain, heart, lungs and kidneys were observed to have more microfilariae in tissue sections. The number of mf seen in the brain and kidneys of animals administered IVM alone tripled that of control animals. Co-administration of IVM + PSE caused a greater increase in mf in the brain and kidneys while the reverse was noticed with the co-administration of IVM + ASA. Conclusions The treatment of Loa hyper-microfilaraemic individuals with ivermectin produces a clinical spectrum that parallels that seen in Loa hyper-microfilaraemic humans treated with ivermectin. The utilization of this experimental model can contribute to the improved management of the adverse responses in humans.

A Photoreactive Fluorescent Marker for Identifying Eosinophils and Their Cytoplasmic Granules in Tissues

Here we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photoreactive complexes that produce stable fluorescent emissions. This method worked readily with histological tissue sections 6–60 μm thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed. Deep red (>605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510–590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm). The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable (>10 min). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore. This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers. Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.

Oncolytic Tanapoxvirus Expressing Interleukin-2 is Capable of Inducing the Regression of Human Melanoma Tumors in the Absence of T Cells

BACKGROUND Oncolytic viruses (OVs), which preferentially infect cancer cells and induce host anti-tumor immune responses, have emerged as an effective melanoma therapy. Tanapoxvirus (TANV), which possesses a large genome and causes mild self-limiting disease in humans, is potentially an ideal OV candidate. Interleukin-2 (IL-2), a T-cell growth factor, plays a critical role in activating T cells, natural killer (NK) cells and macrophages in both the innate and adaptive immune system. OBJECTIVE We aimed to develop a recombinant TANV expressing mouse IL-2 (TANVΔ66R/mIL- 2), replacing the viral thymidine kinase (TK) gene (66R) with the mouse (m) mIL-2 transgene resulting in TANVΔ66R/mIL-2. METHODS Human melanoma tumors were induced in female athymic nude mice by injecting SKMEL- 3 cells subcutaneously. Mice were treated with an intratumoral injection of viruses when the tumor volumes reached 45 ± 4.5 mm3. RESULTS In cell culture, expression of IL-2 attenuated virus replication of not only TANVΔ66R/ mIL-2, but also TANVGFP. It was demonstrated that IL-2 inhibited virus replication through intracellular components and without activating the interferon-signaling pathway. Introduction of mIL-2 into TANV remarkably increased its anti-tumor activity, resulting in a more significant regression than with wild-type (wt) TANV and TANVΔ66R. Histopathological studies showed that extensive cell degeneration with a significantly increased peri-tumor accumulation of mononuclear cells in the tumors treated with TANVΔ66R/mIL-2, compared to wtTANV or TANVΔ66R. CONCLUSION We conclude that TANVΔ66R/mIL-2 is potentially therapeutic for human melanomas in the absence of T cells, and IL-2 expression resulted in an overall increase of therapeutic efficacy.