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.

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.

Analysis of nematode motion using an improved light-scatter based system.

BACKGROUND: The detailed assessment of nematode activity and viability still remains a relatively undeveloped area of biological and medical research. Computer-based approaches to assessing the motility of larger nematode stages have been developed, yet these lack the capability to detect and analyze the more subtle and important characteristics of the motion of nematodes. There is currently a need to improved methods of assessing the viability and health of parasitic worms. METHODS: We describe here a system that converts the motion of nematodes through a light-scattering system into an electrical waveform, and allows for reproducible, and wholly non-subjective, assessment of alterations in motion, as well as estimation of the number of nematode worms of different forms and sizes. Here we have used Brugia sp. microfilariae (L1), infective larvae (L3) and adults, together with the free-living nematode Caenorhabditis elegans. RESULTS: The motion of worms in a small (200 ul) volume can be detected, with the presence of immotile worms not interfering with the readings at practical levels (up to at least 500 L1 /200 ul). Alterations in the frequency of parasite movement following the application of the anti-parasitic drugs, (chloroquine and imatinib); the anti-filarial effect of the latter agent is the first demonstrated here for the first time. This system can also be used to estimate the number of parasites, and shortens the time required to estimate parasites numbers, and eliminates the need for microscopes and trained technicians to provide an estimate of microfilarial sample sizes up to 1000 parasites/ml. Alterations in the form of motion of the worms can also be depicted. CONCLUSIONS: This new instrument, named a "WiggleTron", offers exciting opportunities to further study nematode biology and to aid drug discovery, as well as contributing to a rapid estimate of parasite numbers in various biological samples.

The lack of influence of food and local alcoholic brew on the blood level of Mectizan(®) (ivermectin).

There is concern that extraneous factors, such as food and drink, may alter the pharmacodynamics of Mectizan(®) (ivermectin) in patients receiving this important anti-parasitic drug, and thus might put such individuals in danger of serious adverse events. The effects of a common local alcohol-containing beverage and a local food on plasma levels of ivermectin were studied in Sudanese volunteers after administration of the standard dose used in mass drug administration programs for onchocerciasis and filariasis. Plasma levels of ivermectin at various time points (0-48h) after administration of ivermectin were ascertained by HPLC assay in ten volunteers given 150µgkg(-1) ivermectin together with either a local sorghum-based food ('assida'), or a locally brewed alcoholic beverage ('arangi' made from sorghum grain) or in those who were fasting. Maximum mean (±SD) plasma levels of ivermectin (67±49ngml(-1)) were reached within 2h in fasting patients, and had dropped to 26±20ngml(-1) after 30h. The coadministration of local food or alcoholic beverage did not cause an increase in ivermectin plasma levels above those observed in people who were fasting. However, at 2h after ivermectin administration, patients given alcohol had significantly lower plasma ivermectin levels than fed patients or fasting patients. There were no significant differences among treatments for AUC0-30, Cmax, or tmax, and so the coadministration of local food or alcoholic beverage did not cause any change in pharmacokinetic parameters of ivermectin in the plasma in comparison with fasting. None of the measured levels of plasma ivermectin were greater than those reported in previous studies with this compound. These findings do not support the hypothesis that acute intake of alcohol is an important factor in the development of the serious adverse reactions that can occur during the treatment of loaisis patients with ivermectin (Mectizan(®)).

Transfer of Bacteroides splanchnicus to Odoribacter gen. nov. as Odoribacter splanchnicus comb. nov., and description of Odoribacter denticanis sp. nov., isolated from the crevicular spaces of canine periodontitis patients.

Numerous novel anaerobic bacteria were isolated from the crevicular spaces of dogs with periodontitis. The phenotypic characteristics of these bacterial isolates indicated that they were similar to members of the genus Porphyromonas. However, comparison of the 16S rRNA gene sequences of the isolates indicated that they were related to members of the Bacteroides splanchnicus subgroup. A representative of the novel isolates, strain B106(T), induced alveolar bone loss in a mouse model of experimental periodontal disease. Based on biochemical, morphological, molecular phylogenetic, and pathogenic evidence, it is proposed that the taxonomic subgroup containing these novel isolates and B. splanchnicus should be classified in a new genus, Odoribacter gen. nov., within the family 'Porphyromonadaceae'. In addition, it is proposed that B. splanchnicus should be reclassified as Odoribacter splanchnicus comb. nov., and that the newly identified isolates should be classified as representing Odoribacter denticanis sp. nov., the type strain of which is B106(T) (=ATCC PTA-3625(T)=CNCM I-3225(T)).

The effects of long-term community level treatment with ivermectin (Mectizan) on adult Onchocerca volvulus in Latin America.

The objective of this study was to examine nodules from Mexico, Guatemala, and Ecuador collected over a one-year period (2001) to determine the effects of semi-annual ivermectin treatments on Onchocerca volvulus macrofilarial populations. Nodules were sectioned, stained with hematoxylin and eosin, and histologic findings were compared between countries and with historical data prior to the introduction of ivermectin into the region. Nodules from Ecuador had 10 times more dead or moribund worms than the historical control (66.6% versus 6.5%); nodules from patients from Mexico and Guatemala did not differ from the control. More than 80% of the female worms in each country were uninseminated and producing unfertilized oocytes. Nodules containing males differed in each country from the historical control (P < 0.0001), with presence of males ranging from 19.7% in Mexico to 13.6% in Ecuador versus 73% in the control. Nodules with females producing active microfilariae ranged from 7.8% (Mexico) to 2.7% (Ecuador) versus 60% in the historical control (P < 0.0001). Nodules from Ecuador and Mexico were significantly smaller in size than those from Guatemala or historical controls (P < 0.0005). These results depict a deteriorating condition of adult O. volvulus populations in Mexico, Guatemala and Ecuador, indicating that semi-annual ivermectin treatment of >/=6 years has had a profound effect on survival and reproduction of this species.

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 microm 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.