the initiatives taken to form joint teams and share training work in the Nairobi Mr. E. Manyara, Senior Assistant Director, Trade & Investment. EManyara Investing is located at: Barclays Plaza-Loita Street, Nairobi, Kenya, What is the phone number of EManyara Investing? addition, other viral and bacterial diseases share many of the features Polack, F. P., S. H. Lee, S. Permar, E. Manyara, H. C. Nousari. TURKEY FOR FOREX The give is to densities, words defining patterns the information. It are easy: to of free Zoom or click the the. To is animated Best Internet to.
Indeed, most social-ecological systems have undergone dramatic change in the last century due to climatic, landscape, and institutional shifts. Because coping mechanisms are developed in relation to particular landscapes, livelihoods, and institutions Agrawal, , , social and ecological changes have altered relations across these elements, impacting the effectiveness of particular coping strategies Wangui et al For instance, pastoralists have historically deployed a suite of coping mechanisms in response to the highly variable climate of semi-arid and arid landscapes Barrow et al.
Yet, these capacities maybe increasingly compromised in the rangelands of East Africa due to increasing exposure to climate extremes, such as flood and drought and shifting institutional environments Galvin et al. The mechanisms that pastoralists in East Africa historically utilized to cope with climate variability were part of a tightly coupled system where livelihoods, institutions, and landscapes were mutually reinforcing Fratkin et al.
Pastoralists livelihoods were co-produced with a savanna mosaic landscape managed as a common property system by formal and informal customary institutions Galvin et al. However, these landscapes are undergoing rapid change and fragmentation, with substantial impacts on livelihoods, institutions, and coping capacities Galvin The system has decoupled, with increased diversification and privatization of livelihood practices across a fragmented and compartmentalized landscape, divided into different units managed by various competing institutions, with many key resources enclosed inside farms and conservation areas Homewood et al.
We argue that, as the relationships between livelihoods, landscapes, and institutions change, so do the particular entitlement bundles needed to effectively cope with climatic variability and extreme events. Through a case study looking at responses to a recent drought by Maasai pastoralists in northern Tanzania, we illustrate that pastoralists are adapting to multiple changes and stressors in addition to a changing climate. We show that adaptive capacity is deployed through the use of new techniques to cope with drought within a changing landscape-institutions nexus.
This includes the decline in certain customary strategies e. Coping strategies are differentially adopted by households within a community, reflecting varied and changing asset and entitlement bundles. Entitlements to pasture, historically based on relations of reciprocity, are being replaced by new capabilities of obtaining cash to lease exclusive rights to pasture, and new forms of knowledge and connections to secure such pastures outside Maasai controlled territory.
Such capabilities are only available to some herders. We thus argue that institutional and landscape changes are leading to the emergence of more uneven capacities within and between communities. Additionally, as several of the new strategies further reinforce the very processes undermining pastoral land use in the region-fragmentation, and privatization—we posit that they are not contributing to the overall capacity of the larger social-ecological system to adapt to climate stresses within changing institutional settings.
We present household level data on herders' response to the drought in two Maasai communities in northern Tanzania differentially affected by the event. Maasai refer to this as the most severe drought in living memory, because pastures were severely depleted and mobility occurred on such a large scale. Yet droughts in and resulted in less rainfall, highlighting that vulnerability is not only a function of environmental susceptibility. Our analysis hinges on an appreciation that institutions mediate access to resources and that entitlement and asset bundles enable households to navigate institutions.
This approach may enable increased understanding of adaptive capacity to such events in the future among pastoralists and other societies. Adaptive capacity is a central concept of both vulnerability and adaptation analyses Adger, It is defined as the ability of a system to adapt to climate-related hazards by designing and implementing new strategies or by expanding coping capacity to reduce vulnerability to these hazards Brooks and Adger, We focus on the specific ways in which pastoral communities are modifying coping mechanisms and how such changes reflect longer-term adaptive capacities to cope with climate variability in a fragmented landscape.
While coping mechanisms are often seen as short-term reactions to events Turner et al. Our analysis is not a vulnerability assessment, which would require multiple measurements at various scales of impact. Rather, we use one particular measure of impact during the drought: loss of cattle, the key asset in pastoralist communities, and analyze how it is related to the use of a suite of coping mechanisms. Based on these results, we draw conclusions about the adaptive capacity of different pastoralists to deal with climate variability and extreme events, both predicted to increase with climate change IPCC, Our data capture evidence of evolving drought-coping mechanisms amidst a backdrop of ecological and institutional change, which have increasingly restricted historical coping strategies, namely mobility.
New strategies enable households with the right entitlement bundle e. Our analysis reflects an understanding of adaptive capacity as 1 tied to entitlement and asset bundles, 2 intimately connected to formal, informal; local, trans-local institutions, and 3 scale-dependent. Adaptive capacity depends on the suite of environmental, social, economic, and political entitlements that particular individuals, households, or communities can mobilize to cope with risk Leach, Mearns and Scoones The entitlements approach is most commonly used to discuss vulnerability.
In turn, adaptive capacity is the component of vulnerability best explained by the entitlements approach. The less capacity an individual, community, or society has to adapt to change and variability including extreme events , the more vulnerable they are. Some people have access to certain entitlements while others do not, because of historically contingent processes of social differentiation and development Ribot, These uneven entitlement bundles promote differentiated adaptive capacities among households in a community, and between communities.
It is common for entitlements and endowments to be linked to narrowly defined poverty measurements. This ignores the agency of social units, the role of knowledge and social relations, and the importance of institutions in mediating access to entitlements Turner et al. Agrawal's institutions framework, and his five-fold typology of coping strategies mobility, storage, diversification, communal pooling, and market exchange are particularly useful for understanding the adaptive capacity of changing pastoral systems.
We look at market exchange but focus primarily on mobility, which Agrawal presents as the most natural and common response to climatic variability and stress, particularly for pastoralists Niamir-Fuller, M. Mobility is a key coping mechanism for pastoralists in dealing with seasonal resource variability and drought, with restrictions on mobility negatively affecting adaptive capacity and increasing vulnerability BurnSilver, Worden and Boone , Oba and Lusigi Finally, the scale dependency of adaptive capacity suggests that particular actions or adjustments may produce positive results at one scale, such as the household, while leading to maladaptive outcomes at the larger community or social-ecological system scale Engle Though they can refer to the same scale, the community may be a smaller component of a larger social-ecological system.
Which strategies can be adopted among different households reflects differences in entitlement bundles, providing herders with uneven abilities to navigate institutions across scales. In East Africa, overlapping social institutions historically enabled a suite of coping mechanisms to support pastoralism across semi-arid landscapes where resources are variable in time and space. We now outline the history of this system and the ways in which landscapes and institutions have become fragmented and decoupled, thereby impacting extant coping capacities, particularly mobility.
In this section and the next we draw from historical accounts and the lead author's ethnographic data , living in one of the study villages, six-months in to recount the historical relationships between landscapes and institutions, which mediated Maasai resource use. Maasailand refers to a predominately semi-arid savanna landscape that historically stretched , km 2 from northern Tanzania into southern Kenya, where the rural population is dominated by Maa-speaking people McCabe et al.
Each section was managed as a separate common property system, with social rules directing resource management Galaty, ; Homewood and Rodgers, ; linked to other sections through joint institutional frameworks organized around clan and age-set affiliations Spear and Waller, The age-set and clan links provided formal institutional frameworks for a decentralized system of leadership and informal social relations coordinating access to resources Potkanski, The unit of herding was the polygamous household olmarei, sg; olmareta pl.
More than one olmarei often shared one homestead enclosure boma , Swahili; engang , Maa , with cattle often corralled together at night and sometimes herded together. Pasture access occurred through daily and seasonal mobility see also Butt et al ; Homewood, , , and was coordinated through informal rules within and across olosho boundaries. Reserve pastures a lalili were set aside by groups of households for use by calves and sick cattle.
Relations of reciprocity coordinated access to alalili , water resources also sometimes managed by clans , and pasture. During particularly tough times, no needy herder was denied access to water or pasture. This can be thought of as an extended set of entitlements: long-reaching social relations based on trust and reciprocity. Social networks and good relations with age-mates, clan members, and traditional leaders ilaigwenak were essential for facilitating mobility of livestock across large spatial scales in search of pasture and water Homewood, , Potkanski, , and particularly important during droughts.
As with pastoralists across Africa, mobility was used to optimize the use of forage and reduce risks associated with disease and resource variability across time and space Little et al. Today, while mobility remains an important herd management strategy, it has been constrained by fragmentation and conflicting institutional arrangements, with much of Maasailand now inaccessible to many if not all Maasai.
The tightly coupled system has fragmented and decoupled. The country has changed. Long ago, we moved like the wildebeest. Fragmentation has been noted as the single largest factor impacting pastoral rangelands today Galvin et al, a ; Hobbs et al. In this section, we describe the ways in which fragmentation is occurring in Maasai rangelands covering all three types discussed by Hobbs et al , : dissection, conversion, and compression.
Landscapes are dissected through the creation of physical, social, and administrative boundaries; conversion turns pieces of rangeland into croplands or protected areas; and compression occurs with sedentarization and the increased density of people and animals in remaining rangelands see also Fratkin, Fragmentation disconnects and dissects heterogeneous landscapes, constraining the mobility of people and animals and restricting their access to necessary pasture and water Hobbs et al, The creation of villages created new units of administration and new parameters for settlement, affecting the ways in which people thought about and practiced mobility Parkipuny This is expressed by a group of women in one of the study villages when asked when mobility patterns changed Group interview, Oltukai village :.
When the Makaa age-set started as warriors and when Operation Imparnati started to cut up areas for people to stay. People started to get places to stay. Before this, people would move. The elders would say let's go, all of Oltukai, let's move. It is not like this anymore, because we have come to get villages. If we leave to go to Mswakini [another village] we can't because other people live there and now we have got our own place to stay.
Villages are administered by an elected chairman, council members, and a salaried executive officer. Most villages are too small to encompass all the resources needed by pastoralists throughout the year, especially during the early rains which vary considerably across space and during droughts. Mobility still occurs as a part of customary herd management, and is intensified during drought, often taking people and animals beyond village boundaries in search of water and pasture.
Yet as expressed by the women in the quote above, today rather than entire families moving, young men often move with cattle to temporary bomas ronjo , leaving women, children and elders behind in the resident village to attend school, protect farms, and attend to small stock.
With increased sedentarization and farming, more land within Maasai villages is being privatized and converted to cropland Homewood, et al, ; McCabe, et al. This means that mobility both within and beyond village boundaries is complicated by property delineations and conversion. There has also been a trend towards smaller land units, the individualization of settlements and livestock management, and the titling of land for tenure security Homewood et.
Customary formal and informal institutions are giving way to village and regional governance structures and private household heads in regulating access to resources Cooke, ; Goldman, Much land has been converted into large-scale commercial farms and conservation areas, including parts of Tarangire, Arusha, and Serengeti National Parks, Ngorongoro Conservation Area, Lake Burungi and Enduimet WMAs, Manyara Ranch, and Mkomazi Game Reserve, which enclose drought reserve pastures and water sources for exclusive use by wildlife Igoe, Livelihood changes have also contributed to reduced mobility, while also increasing wealth stratification within Maasai societies Homewood et al Those with the right bundle of entitlements cultivate large plots and intensify for-market livestock production using improved breeds.
With more children in school, and young men working in cities, many families are often left without the labor needed to move cattle long distances Goldman There is also an increased privatization of land and livelihoods with an associated decline in social pooling of resources and labor Goldman, and field notes All these changes pose a growing challenge to the use of mobility on a regular seasonal basis as a resource management tool. Yet, as mentioned above, mobility is still practiced, if in new and different ways.
In doing so, herders drew on remaining customary institutional frameworks while also negotiating with village government leaders, national park personnel, and private landowners in ways that required new forms of social relations, knowledge, and skills. Looking at data from collected in two related regions differentially impacted by the drought, we show that these new arrangements and capacities are needed to practice pastoralism across a fragmented landscape with conflicting institutional frameworks.
Research was conducted in two administrative districts: Monduli and Longido, in the Kisongo Maasai olosho. Data come from specific areas recognized as sub-sections within each district, Emanyara and Longido Figure 1. Compared to Emanyara, Longido is slightly more arid, and was more severely impacted by the drought: was the third straight year of little-to-no rainfall. For this reason, unusually large numbers of herders from Longido migrated to Emanyara in the latter was relatively better off, having received some rainfall early in the season.
Site selection was influenced by the lead authors long-term research in Emanyara and the large number of migrants to this area from Longido during the drought. These sites capture different impacts of the drought while exhibiting common patterns of landscape fragmentation and livelihood practices for Maasailand. Both study sites reflect the constraints of an increasingly fragmented landscape Homewood et al.
The villages in Emanyara, Oltukai and Esilalei, used to be part of one larger village that ran up to a peri-urban center bordering Lake Manyara National Park LMNP , where herders accessed fresh water springs in the dry season. These springs were taken over by farms, and the larger village split into three smaller ones Goldman, Esilalei also borders Manyara Ranch and LMNP, and is at risk of losing dry season grazing areas to new conservation units in the eastern highlands Goldman, The number of farms more than doubled in both villages between and Goldman, Individuals have begun to privatize their alalili reserve pastures or set them up exclusively for sub-village access.
Residents of both villages used to regularly move cattle to Maramboi , a wooded pasture on the southern edge of Lake Manyara, at the start of the rains and during droughts, but the creation of the Lake Burunge WMA in prohibited grazing there. Grazing inside Manyara Ranch is allowed on a limited basis, by village residents in the dry season only, and is enforced by game scouts Goldman, Important dry season reserves and water sources inside Tarangire National Park became off-limits with the establishment of the park in the s Igoe, Discussions of creating conservation corridors in the area raises the risk of the removal of more grazing land for conservation Goldman, Longido district has also been divided in many ways for conservation, growing agricultural development, and a new administrative center.
Longido district is bounded by conservation areas: Ngorongoro Conservation Area to the west, Kilimanjaro and Arusha National Parks to the east and south, and Amboseli National Park just across the Kenyan border to the north Trench et al. The Enduimet WMA, where grazing is prohibited, was established in as a pilot project and today straddles land in eight villages Benjaminsen et al.
There has also been a steady increase in farming by Maasai and non-Maasai throughout the area, further fragmenting the land within Maasai control Trench et al. At the end of the drought January - February , structured interviews were conducted across the two areas: 51 in Emanyara and 97 in Longido.
The larger sample for Longido is related to it being larger in size and population and more impacted by the drought. Our estimated average losses for both areas are consistent with estimates reported in a follow up study , and from reports of organizations working in the area. We used these data to look at how the effect of the drought on livestock holdings was mediated by different coping mechanisms, money spent, and site. The impact of the drought was measured by the outcome of cattle loss, or the ratio of cattle that died during the drought to the total pre-drought holdings.
This is consistent with other work measuring the impact of drought among pastoral societies McPeakand Barrett, Our unit of analysis is the household, measured through the individual herder in charge of livestock, generally the household head.
Household level analyses are common in pastoral areas, where a household is usually defined as a male-headed polygamous unit, with multiple dependents and potential contributors to household entitlements salary, labor, education, and connections Homewood, We interviewed those in charge of managing livestock and making decisions about mobility and overall livestock care e. The contributions of other household members money and labor were addressed in particular questions regarding where money came from and what restricted certain activities.
While there are clearly some limitations to this approach, it was necessary when interviewing people away from home, and does reflect the entitlements of the entire household through questions on contributions. Insights regarding adaptive capacity were gained by examining the suite of coping mechanisms used by households. In addition to looking at the amount of money spent in coping activities, we asked whether households bought pumba a mixture of grain shafts and corn husks , illegally grazed inside national parks or on private property, purchased exclusive grazing rights on private land, split herds, sold cattle, or purchased veterinary medicine.
Illegal grazing was removed from the quantitative analyses because of concern over accuracy of the data but is addressed in the ethnographic data. To assess the role of a particular coping strategy, we estimated Poisson regressions on the number of cattle lost. By adding the pre-drought holdings as an offset, the estimation is mathematically equivalent to modeling the rate of loss Powers and Xie : , and as such appropriate to studying cattle mortality risks e.
The exponentiated coefficients of the model are risk ratios, the relative difference in the rate of cattle lost from adopting vs. In addition to the survey data, in-depth ethnographic data were collected at the beginning March-July and after February the drought. This included unstructured interviews with herders in Emanyara, including migrant herders from Longido, observation of particular individuals and casual conversations through participant and non-participant observation.
These data were used to expand findings from the survey, which we first describe on the aggregate with our multivariate analyses and then more detail using descriptive analyses. All names used in the manuscript are pseudonyms. He can be seen as representative of the larger population in that his herd size is slightly above the overall average , but below the Emanyara average and he is recognized as an average player in the village in terms of politics and livelihood.
The increased severity of the drought in Longido may have limited which coping strategies were effective for herders in the area, thus compounding its effects. For instance mobility and the sale of cattle often occurred after animals were already in poor condition, thus bringing little relief. Finally, five herders in Longido and 1 in Emanyara reported using money from a business and six herders all from Longido reported receiving money from a son who worked in town.
Although these results suggest that a diversity of sources were used, note they do not denote the percentage of money coming from each source. Money was spent in different ways in the two sites, as illustrated in Panel B, Table 2. Herders in Longido needed to move their cattle longer distances and multiple times and thus spent a lot of money to carry small calves, scout for grass and water, purchase food and fuel, and for networking i.
These results are further illustrated in our multivariate Poisson model see Table 3 , Model I , where being from Longido is associated with a rate of loss 3. Instead, our results suggest that a significant investment on specific strategies was required to avert high loss levels.
Some strategies were not effective at all and might have even been counterproductive while drawing on resources. For instance, herders buying pumba had a considerably higher loss ratio than the very few not doing so. Concentrating resources by selling cattle is an important coping strategy if done early, before cattle are too thin and the markets too saturated with stock McPeak and Little, Our results also illustrate that several types of mobility requiring a substantial investment were key in reducing loss.
This practice involved paying a landowner outside of the village for exclusive rights to graze on their land, often in different micro-climatic zones e. Those with large herds likely had the necessary bundle of entitlements access to cash, knowledge and social connections in Maasai and non-Maasai areas to enact the strategies that worked best.
Consistent with this notion, moving cattle and buying pasture were particularly relevant for this group. This lack of statistical significance is unlikely to be the result of low statistical power as the sample size of the two groups is similar.
Indeed, those with smaller herds likely had access to fewer entitlements and assets and thus employed fewer coping strategies, which rendered them more vulnerable to the drought. As the drought was more severe in Longido than Emanyara, this suggests that in worse environmental conditions, those with fewer assets and entitlements are more vulnerable.
In our post-drought interviews, herders were asked to reflect on their ability to protect their cattle during the drought. Only 17 individuals thought they did well, all from Emanyara. Of these, 6 stated they were able to move early—before the drought worsened and before others arrived at a new location. Perhaps most interesting is that 10 of the 17 herders stated that they did well because they had an alalili they were able to use, with two of these individuals stating that their success was based on denying others access to these reserve pastures, a violation of Maasai norms of reciprocity.
An alalili is often managed by a boma , or a group of bomas , with access available to others as long as the grazing is for calves or sick cows. During the drought, access to alalili pastures was denied for the first time herders in Emanyara could recall, which reflects another result of the privatization of management.
Of those who thought they fared poorly during the drought, 78 stated the severity of the drought and lack of money as the reason. People also mentioned spending more money then ever before on veterinary medicine. This was likely related to moving cattle to places they had never moved to before. We now present ethnographic data of a couple of individuals whose experiences are typical of those who did well in this drought.
Their experience demonstrates how access to an entitlement bundle of cash, social networks, and knowledge on how to interact with new institutional arrangements were strategic in mitigating risk during this drought in new ways. In Emanyara, at early signs of the drought in November-December, a few herders moved their cattle to pastures in Simanjiro district—which had received more rainfall and had more open pastureland.
Their ability to do so was based on connections in the area; access to cash to buy veterinary medicine to protect against tick and tsetse-fly borne diseases more prevalent in Simanjiro; a car to survey pastures and carry calves; and the ability to pool labor with other herders. Not everyone was able to move at this early stage.
Rains eventually came to Emanyara and village government officials in Simanjiro sent migrant herders back home, though those from places that did not receive rain e. In this way, local government institutions acted to mitigate grazing practices historically negotiated along customary lines. During the long rains in March-May, Oltukai village was one of the few villages to receive several days of rainfall and have a dam filled with water.
It was thus subjected to large numbers of in-migrating herds, particularly from Longido District but also from Kenya. As the rains ended early and pastures were nearly depleted due to the increased number of migrant cattle, those with the ability to do so began to look for places to move their herds.
As rainfall was sporadic, options were few and herders began to look outside the bounds of their customary movement patterns, drawing on new and different social networks. The story of Maika and his friends is particularly revealing. As migrant herders moved into his village, Maika took advantage of this opportunity to exchange information, make new friends, and link into larger social networks.
He befriended Mollel, a wealthy herd-owner from Longido, who was looking for a place to move his cattle. Maika and Mollel complemented each other well. Mollel had money and a car, allowing them to set off on a weeklong tour in search of pasture. Maika, who grew up in the area, had connections with local Maasai and many non-Maasai in surrounding places as he had previously been involved in marketing maize and was currently involved in the cattle market business.
Maika's connections proved invaluable in purchasing grazing rights in the nearby agricultural town of Karatu. With this, Maika moved half his herd to this pasture until the near end of the drought. When pasture was nearly depleted in Oltukai and neighboring Manyara Ranch, Maika once again set off in search of pasture for the remainder of his herd. Magugu is an irrigated area and still had water and grass when other areas were dry. Cattle were fed in farms during the day and snuck into Tarangire National Park at night.
This cost additional money and Maika sold 80 goats to have cash on hand to pay fines when caught grazing inside the park, which happened several times. He also had a motorcycle, enabling him to move between split herds and return home if needed. Maika benefitted from the help of a younger brother in charge of cattle in one location far from home while pooling labor with other herders elsewhere.
Eventually the pasture in Karatu was depleted and he relocated those cattle to Magugu as well. Maika returned home with his cattle on November 17, , after several days of rainfall. On the way home with his cattle he paid a fine for grazing inside the WMA. Maika's success during this drought came from his ability to combine old and new sources of knowledge, skills, and capital, and act in a timely manner.
Each marker indicates data from an individual mouse, with group means represented by a horizontal bar. Data are combined from five of five identical experiments, each showing similar results. Each marker indicates data from an individual mouse, with median values represented by a horizontal bar. Data are combined from two of two identical experiments showing similar results.
Additional booster vaccinations were given at day 21, with or without electroporation. DOM-antigen fusion vaccine design. Schematic diagram indicating DNA fusion vaccine design. The control vaccine p. Each vaccine includes a leader sequence at the amino terminus. Wt A and gag-Tg B mice were immunized with p. DOM-gag or p. Test samples were set up in triplicate; baseline responses without peptides are indicated. Representative data from 1 of 2 identical experiments are shown.
The number n of animals pooled per group is indicated. Wt and gag-Tg mice were immunized with p. A Representative data from individual mice. DOM-gag on days 0 and 28; booster injections at day 28 were administered with electroporation. Representative data from one of 2 identical experiments are shown.
Gag-Tg mice were immunized with p. Coded specimens were analyzed for inflammation and lymphocyte infiltration by a liver pathologist in a blinded manner. All portal tracts were normal, with no expansion and normal biliary structures. There was no inflammation in portal tracts or parenchyma.
There was no hepatocyte degeneration or loss, and the normal sinusoidal architecture was present. Representative sections from gag-Tg mice immunized with p. DOM DNA vaccines on days 0 and 28; booster injections at day 28 were administered with electroporation.
At day 36, splenocytes from individual mice were cultured with 0. No lytic activity was detectable in splenocyte cultures from mice vaccinated with the control vaccine p. Representative data from individual mice are shown; data from one of two identical experiments are shown. Error bars: standard error of the mean. B The ratio of gag to control peptide-pulsed cells surviving in each recipient was calculated.
The ratios were normalized, by assuming that the mean ratio was in mice given the control vaccine p. DOM , and pooled to calculate the mean proportion of surviving donor cells pulsed with the gag peptide, together with the standard error of the mean.
Data are pooled from two of two identical experiments, each giving similar results; the number n of animals pooled per group is indicated. At day 36 mice were challenged by i. Survival data in each panel A, B are pooled from two of two identical experiments, each giving similar results.
The H-2L d -restricted gp70 epitope AH1 has been described previously Construction of the DNA fusion vaccine p. DOM-AH1 has been described The p. DOM control vaccine encodes the first domain of FrC alone. This was used as a template to construct p.
Zhu University of Southampton, Southhampton, U. Briefly, C H 3-encoding sequence was cut from p. The resulting PCR product was gel purified, digested, and cloned into p. BCL 1 in the place of CH 3 , creating p. All injections were administered using a 26 G needle. Animal welfare and experimentation were conducted in accordance with local Ethical Committee and United Kingdom Coordinating Committee for Cancer Research guidelines, under Home Office license.
The skin overlying the quadriceps muscle was shaved, and DNA vaccine was administered using the indicated dose and volume. Following the application of a conductance gel, silver electrodes were placed on the skin on either side of the injection site and a local electrical field was immediately applied using a custom-made pulse generator, Elgen Inovio , as previously described During tumor challenge, mice were injected s.
For prophylacetic immunization, mice were challenged with tumor cells 14 days after DNA vaccination, while for therapeutic immunization tumor cells were injected 1 day before DNA vaccination. All mice were monitored twice daily for tumor development and were culled when mean tumor diameter reached 15 mm, in accordance with humane end point guidelines United Kingdom Coordinating Committee for Cancer Research. To monitor priming of humoral immunity, mice were vaccinated i.
The injection schedule day 0, day 21 follows the previously published protocol for Ab induction, established using Id IgM protein vaccination For protein vaccinations, Id IgM from BCL 1 was coupled to FrC protein using a one-step glutaraldehyde method, as used for coupling to keyhole limpet hemocyanin Serum IgG titers were compared using a two-tailed t test on log normalized data.
The inventors tested the ability of the p. A second experiment confirmed this trend FIG. Responses to the control vaccine p. DOM were insignificant. The effect of injection volume on immune outcome was confirmed in a second experiment FIG. Again, functional activity was confirmed by cytotoxicity assay following a 6-day expansion in vitro FIG. The inventors next assessed the effects of combining DNA vaccine delivery with electroporation. Results FIG.
Three experiments were conducted to confirm this point, because experiment 1 showed a decrease in response when using electroporation. However, the additional two experiments showed no change, and data compiled from the three identical experiments indicated no effect of electroporation using this optimized volume FIG.
The inventors have previously demonstrated that following vaccination with p. To assess therapeutic efficacy, the inventors investigated the effects Of DNA vaccination 1 day after tumor injection. The effects of electroporation on priming were clear only when using suboptimal vaccination conditions. The inventors then investigated whether electroporation could improve performance of optimal delivery when combined with boosting.
Electroporation was given either at priming alone, at boosting alone, or at both time points. DOM-AH1 at day 0 only without electroporation were low mean 0. Electroporation at both the priming and boosting stages was also effective in amplifying the response to naked DNA mean 2.
This vaccine is known to induce significant levels of Ab against both tumor-derived idiotypic Ig and FrC components of the fusion gene, and this is confirmed in FIG. A single injection of the p. Electroporation at priming increased anti-Id Ab at both time points. By way of exemplification, FIG. Mice were vaccinated at days 0 and 28 with DNA vaccines day 28 injections given with electroporation to aid delivery.
DOM-antigen is from an infectious disease, demonstrating the applicability of this approach against both cancer antigens and other diseases. In this case, it is the idiotype scFv from 5T33 murine myeloma. Mice were vaccinated at days 0 and 21 and 42 with DNA vaccines. The mice were bled at day 35 to assess anti-5T33 myeloma cancer cells at day 63 and survival monitored. Anti-5T33 antibody responses were detected A. Vaccine clearly protects from 5T33 cancer B.
These results demonstrate that p. DOM-antigen vaccine design can induce disease-specific antibody response. In this case the target antigen if from a tumor cell. There are two major problems in developing DNA vaccination as a treatment for cancer. The first is the poor immunogenicity of most candidate tumor Ags.
There are many strategies aimed to increase this 20 , and we have chosen to use fusion genes that encode tumor Ags in combination with immunogenic pathogen derived sequences, mainly derived from tetanus toxin Different designs have been optimized to induce effector pathways for precision attack on tumor targets Currently, the inventors are testing these in clinical trials, with early evidence for immune responses.
The second problem, relevant for all DNA vaccines, relates to the translation of promising data in animal models to human subjects. Although safety does not appear to be an issue, the efficacy in humans has been disappointing , partly due to difficulties in scaling up DNA vaccine dose and injection volume for human application Cellular uptake of DNA appears to be a significant limiting factor on transfection in vivo, and low vaccine dose results in poor Ag expression and reduced immunogenicity Similarly, injection volume can influence Ag expression and immunogenicity in vivo Hydrostatic pressure created by a relatively large injection volume into a small muscle may distend the extracellular space between muscle cells and facilitate the transfer of macromolecules across the plasma membrane This effect will be reduced in large animals and humans, because the ratio of injection volume to muscle mass is far lower In vivo electroporation can increase DNA uptake by muscle cells and mononuclear cells at the site of injection 27, 44 , leading to increased Ag expression Dendritic cells at the draining lymph nodes have been shown to contain DNA originating from the injection site 26 , and electroporation might also contribute an undefined adjuvant effect, possibly mediated through local tissue damage and release of inflammatory factors The inventor's murine data confirm that induction of antitumor CTL by DNA fusion vaccines is dependent on dose and volume of injection 26, However, induction of Ab appears far from optimal under the same conditions and electroporation amplifies priming significantly.
This could reflect a need for higher levels of Ag for priming of Ab responses 47, Electroporation therefore offers a strategy to amplify priming, which could be useful in the clinic. The amplification is reminiscent of that achieved by boosting with Ags delivered via viral vectors These vectors are presumed both to increase protein expression and to stimulate an inflammatory response 50, Their disadvantages, particularly for cancer patients, are that pre-existing or developing immunity can neutralize the delivery agent and negate continued use A more general disadvantage is that highly immunogenic viral or bacterial vectors may introduce potentially immunodominant T cell epitopes, possibly out-competing weakly immunogenic tumor Ags in the ensuing immune response The mechanism by which electroporation amplifies CTL or Ab responses when administered at the stage of boosting is unclear.
For Ab induction, in addition to a more effective induction of T cell help, more available Ag would be provided on boosting for uptake by B cells 47, 48, Electroporation also leads to an inflammatory response, which is likely to recruit specific T and B cells to the injection site This turned suboptimal delivery for CTL induction into effective vaccination and should be translatable to human subjects. Electroporation devices are now acceptable for human subjects 60 and have already been tested in volunteers The inventors have started a clinical trial in patients using the same device Protocols for electrical stimulation have to balance immune outcome with patient acceptability, and further trials in large animals and patients will assist optimization.
This simple modification should facilitate application to the clinic. Construction of a DNA vaccine p. DOM vaccine was then used as a template to construct p. Fusion to the C-terminus gives optimum processing and presentation. Vaccine integrity was confirmed by DNA sequencing.
The B6 gag-transgenic model, in which the gag protein from FMuLV is expressed under the control of the mouse albumin promoter in the liver, has been described previously. Triplicate sample wells were tested with a range of gag peptide concentrations; control samples were incubated without peptide.
Peptide-specific ELISpot responses greater than 60 spot forming cells SFC per million splenocytes and more than twice baseline values observed in the absence of peptide were considered positive. The data were then pooled within each experimental group to calculate the mean ELISpot response as a percentage of the maximum observed response for each peptide concentration.
To assess gag -specific CTL responses, vaccinated mice were sacrificed at day 36 and their spleens were removed. Single cell suspensions were made from individual spleens in complete medium. All mice were monitored daily and were euthanized on detection of tumor development, in accordance with humane end point guidelines UKCCCR. Following priming day 0 and booster vaccinations day 28 groups of B6 and gag-Tg mice were euthanized on day 36 to assess autoimmune injury.
Experimental groups were compared using an unpaired, two-tailed t test. Survival curves were compared using the Chi square log-rank test. The ability of the p. Vaccination with p. The control DNA vaccine p. Gag-Tg mice were also tested for their ability to respond to the p. The control p. A survey of larger numbers of individual mice was then carried out, which demonstrated that although p. Wt and gag-Tg mice were vaccinated with either p. To assess this, blood was drawn from gag-Tg mice at day 36 following vaccination and serum levels of the liver enzymes AST and ALT measured as indicators of liver injury.
In addition, mice were sacrificed at this time point for blinded histological analysis of liver tissue. DOM-gag DNA vaccine in gag-Tg mice to exhibit lytic activity against targets expressing the gag epitope was tested. At day 36 after vaccination, splenocytes from wt or gag-Tg mice were stimulated in vitro with peptide for 6 days and lytic activity assessed in a 51 Cr release assay.
CTL from either wt or gag-Tg mice, primed with p. No specific CTL activity was generated by culture of splenocytes in vitro with gag peptide following vaccination with the control vaccine, p. To address this, wt and gag-Tg mice were vaccinated with either p. At day 36, mice were injected intravenously with sex-matched, syngeneic splenocyte targets pulsed with either gag peptide or control peptide that had been differentially labeled with CFSE to permit distinction between the two targets by flow cytometry.
Wt mice that had previously been vaccinated with either p. Notably, in gag-Tg mice vaccinated with p. DOM-gag, despite the absence of ongoing liver toxicity, the gag peptide-pulsed target cells were similarly eliminated in a peptide-specific manner FIG.
The degree of specific target cell lysis did not differ significantly between wt and gag-Tg strains immunized with this DNA vaccine FIG. This represents the typical challenge that might be anticipated for targeting human tumor-associated antigens, in which the candidate antigen is detected in normal tissues but over-expressed in the malignancy. To address this, wt or gag-Tg mice were vaccinated with p.
DOM-gag or the control vaccine p. Immunization with p. DOM , suggesting that injection of this dose of FBL-3 leukemia cells alone can lead to the spontaneous induction of natural protective immunity in wt mice FIG. In these mice, there was no evidence for spontaneous induction of immunity following injection of FBL-3 cells, since all naive or control vaccinated mice succumbed by day 19 FIG. Protection by the p. The majority of known human tumor-associated antigens derive from non-mutated self-proteins.
T-cell tolerance, essential to prevent autoimmunity, must therefore be cautiously circumvented to generate cytotoxic T-cell responses against these targets. Candidate MHC Class I-binding tumor peptide sequences are fused to the C-terminus for optimal processing and presentation. The single epitope design allows a focused CTL response, reducing the risk of cross-reactive autoimmunity. However, targeting several epitopes derived from the same or an alternative antigen would be advantageous and would reduce the likelihood of tumor escape due to antigenic mutation or deletion.
To avoid immunodominance effects, delivery of the second vaccine could be into a separate site. An integrated attack on multiple epitopes expressed by leukemic cells could compensate for the loss of antigen-specific T cell frequency and avidity observed in this tolerized repertoire and improve survival.
To model performance against a leukemia-associated antigen in a tolerized setting, we constructed a fusion vaccine encoding an immunodominant CTL epitope derived from Friend Murine Leukemia Virus gag protein FMuLV gag and vaccinated tolerant FMuLV gag -transgenic mice. These results demonstrate a simple strategy to engage anti-microbial T-cell help to activate polyclonal lower avidity but still leukemia-reactive CTL from a tolerized repertoire.
Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
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An investor may experience losses due to factors affecting the overall performance of financial markets. Stock market bubbles and crashes are good examples of heightened market risk. You can, however, hedge against market risk. Even though systematic risk affects the entire stock market, the extent to which the market feels the impact can be minimized. Changes in purchasing power due to inflation may cause inflation risk. Treasury inflation-protected securities to minimize inflation risk.
You can minimize this risk to a good extent by diversifying. A good option is index investing where risk is diversified over the various stocks held in a portfolio tracking a particular index. They invest heavily in stable large-cap U. Continue to Part 3. Part 1 - Why investors prefer dividends in a low interest rate environment. Part 3 - Must-know: Dividends as a hedge against stock market bubbles. Part 4 - Why dividend ETFs may be the right thing at the right time.
The Juneteenth holiday weekend may come as a bit of respite for investors. Last week, they had to navigate increasingly turbulent markets: The officially entered a bear market on Monday, the Federal Reserve announced a 0. Is the Stock Market Closed on Juneteenth?
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Now for the fun part: digging into the top marijuana companies. You might also want to check out marijuana-focused exchange-traded funds ETFs. Below is a list of top marijuana stocks to thoroughly consider. Investing in marijuana companies is not suitable for everyone. For some, particularly conservative investors, the best approach is to avoid these types of stocks entirely.
Only investors who understand and can tolerate high levels of risk should add cannabis companies to their portfolios. Even for aggressive investors, putting too much of your investment portfolio into any one marijuana stock or ETF isn't wise. Consider starting with a small position in a marijuana stock and adding to your holdings as the cannabis market grows and the company increases its revenue and earnings.
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Some changes -- such as if the U. Tremendous growth is likely for the global marijuana industry, but it may not occur evenly or predictably.