A costing exercise of provision of prevention of HIV transmission from mother to child services in Vietnam

Objectives. This dissertation focuses on estimating the cost of providing a minimum package of prevention of mother-to-child HIV transmission (PMTCT) in Vietnam from a societal perspective and discussing the issues of scaling-up the minimum package nationwide. ^ Methods. Through collection of cost-related data of PMTCT services at 22 PMTCT sites in 5 provinces (Hanoi, Quang Ninh, Thai Nguyen, Hochiminh City, and An Giang) in Vietnam, the research investigates the item cost of each service in minimum PMTCT packages and the actual cost per PMTCT site at different organizational levels including central, provincial, and district. Next, the actual cost per site at each organizational level is standardized by adjusting for HIV prevalence rate to arrive at standardized costs per site. This study then uses the standardized costs per site to project, by different scenarios, the total cost to scale-up the PMTCT program in Vietnam. ^ Results. The cost for HIV tests, infant formula, and salary of health workers are consistently found to be the biggest expenditures in the PMTCT minimum package program across all organizational levels. Annual cost for drugs for prophylaxis treatment, operating and capital, and training costs are not substantial (less than 5% of total costs at all levels). The actual annual estimated cost for a PMTCT site at the central level is nearly VND 1.9 billion or US$ 107,650 (exchange rate US$ 1 = VND 17,500) while the annual cost for a provincial site is VND 375 million or US$ 21,400. The annual cost for a district site is VND 139 million (∼US$ 8,000). ^ The estimated total annual cost to roll out the PMTCT minimum package to the 5 studied provinces is approximately US$ 1.1 million. If the PMTCT program is to be scaled-up to 14 provinces until 2008 and up to 40 provinces through the end of 2010 as planned by the Ministry of Health, it would cost the health system an approximate annual amount of US$ 2.1 million and US$ 5.04 million, respectively. The annual cost for scaling-up the PMTCT minimum package nationwide is around US$ 7.6 million. Meanwhile, the total annual cost to implement PMTCT minimum packages to achieve PMTCT national targets in 2010 (providing counseling service to 90% of all pregnant women; 60% of them will receive HIV tests and 100% of HIV (+) mother and their newborn will receive prophylaxis treatment) would be US$ 6.1 million. ^ Recommendations. This study recommends: (1) the Ministry of Health of Vietnam should adjust its short-term national targets to a more feasible and achievable level given the current level of available resources; (2) a detailed budget for scaling-up the PMTCT program should be developed together with the national PMTCT action plan; (3) the PMTCT scaling-up plan developed by the Ministry of Health should focus on coverage of high prevalence population and quality of services provided rather than number of physical provinces reached; (4) exclusive breastfeeding strategy should be promoted as part of the PMTCT program; and (5) for a smooth and effective rolling out of PMTCT services nationwide, development of a national training plan and execution of this plan must precede any other initiations of the PMTCT scaling-up plan. ^

THE UBIQUITIN LIGASE UBE4B IS REQUIRED FOR EFFICIENT EPIDERMAL GROWTH FACTOR RECEPTOR DEGRADATION

The length of time that integral membrane proteins reside on the plasma membrane is regulated by endocytosis, a process that can inactivate these proteins by removing them from the membrane and may ultimately result in their degradation. Proteins are internalized and pass through multiple distinct intracellular compartments where targeting decisions determine their fate. Membrane proteins initially enter early endosomes, and subsequently late endosomes/multivesicular bodies (MVBs), before being degraded in the lysosome. The MVB is a subset of late endosomes characterized by the appearance of small vesicles in its luminal compartment. These vesicles contain cargo proteins sorted from the limiting membrane of the MVB. Proteins not sorted into luminal vesicles remain on the MVB membrane, from where they may be recycled back to the plasma membrane. In the case of receptor tyrosine kinases (RTKs), such as epidermal growth factor (EGF) receptor, this important sorting step determines whether a protein returns to the surface to participate in signaling, or whether its signaling properties are inactivated through its degradation in the lysosome. Hrs is a protein that resides on endosomes and is known to recruit sorting complexes that are vital to this sorting step. These sorting complexes are believed to recognize ubiquitin as sorting signals. However, the link between MVB sorting machinery and the ubiquitination machinery is not known. Recently, Hrs was shown to recruit and bind an E3 ubiquitin ligase, UBE4B, to endosomes. In an assay that is able to measure cargo movement, the disruption of the Hrs-UBE4B interaction showed impaired sorting of EGF receptor into MVBs. My hypothesis is that UBE4B may be the connection between MVB sorting and ubiquitination. This study addresses the role of UBE4B in the trafficking and ubiquitination of EGF receptor. I created stable cell lines that either overexpresses UBE4B or expresses a UBE4B with no ligase activity. Levels of EGF receptor were analyzed after certain periods of ligand-induced receptor internalization. I observed that higher expression levels of UBE4B correspond to increased degradation of EGF receptor. In an in vitro ubiquitination assay, I also determined that UBE4B mediates the ubiquitination of EGF receptor. These data suggest that UBE4B is required for EGFR degradation specifically because it ubiquitinates the receptor allowing it to be sorted into the internal vesicles of MVBs and subsequently degraded in lysosomes.

Bifurcation and singularity analysis of a molecular network for the induction of long-term memory.

Withdrawal reflexes of the mollusk Aplysia exhibit sensitization, a simple form of long-term memory (LTM). Sensitization is due, in part, to long-term facilitation (LTF) of sensorimotor neuron synapses. LTF is induced by the modulatory actions of serotonin (5-HT). Pettigrew et al. developed a computational model of the nonlinear intracellular signaling and gene network that underlies the induction of 5-HT-induced LTF. The model simulated empirical observations that repeated applications of 5-HT induce persistent activation of protein kinase A (PKA) and that this persistent activation requires a suprathreshold exposure of 5-HT. This study extends the analysis of the Pettigrew model by applying bifurcation analysis, singularity theory, and numerical simulation. Using singularity theory, classification diagrams of parameter space were constructed, identifying regions with qualitatively different steady-state behaviors. The graphical representation of these regions illustrates the robustness of these regions to changes in model parameters. Because persistent protein kinase A (PKA) activity correlates with Aplysia LTM, the analysis focuses on a positive feedback loop in the model that tends to maintain PKA activity. In this loop, PKA phosphorylates a transcription factor (TF-1), thereby increasing the expression of an ubiquitin hydrolase (Ap-Uch). Ap-Uch then acts to increase PKA activity, closing the loop. This positive feedback loop manifests multiple, coexisting steady states, or multiplicity, which provides a mechanism for a bistable switch in PKA activity. After the removal of 5-HT, the PKA activity either returns to its basal level (reversible switch) or remains at a high level (irreversible switch). Such an irreversible switch might be a mechanism that contributes to the persistence of LTM. The classification diagrams also identify parameters and processes that might be manipulated, perhaps pharmacologically, to enhance the induction of memory. Rational drug design, to affect complex processes such as memory formation, can benefit from this type of analysis.

Specific, Reversible Cytostatic Protection of Normal Cells Against Negative Effects of Chemotherapy

Chemotherapy is a common and effective method to treat many forms of cancer. However, treatment of cancer with chemotherapy has severe side effects which often limit the doses of therapy administered. Because some cancer chemotherapeutics target proliferating cells and tissues, all dividing cells, whether normal or tumor, are affected. Cell culture studies have demonstrated that UCN-01 is able to reversibly and selectively arrest normal dividing cells; tumor cells lines do not undergo this temporary arrest. Following UCN-01 treatment, normal cells displayed a 50-fold increase in IC50 for camptothecin; tumor cells showed no such increased tolerance. We have examined the response of the proliferating tissues of the mouse to UCN- 01 treatment, using the small bowel epithelium as a model system. Our results indicate that UCN-01 treatment can cause a cell cycle arrest in the gut epithelium, beginning 24 hours following UCN-01 administration, with cell proliferation remaining suppressed for one week. Two weeks post-UCN-01 treatment the rate of proliferation returns to normal levels. 5-FU administered during this period demonstrates that UCN-01 is able to provide protection to normal cells of the mouse within a narrow window of efficacy, from three to five days post-UCN-01. UCN-01 pretreated mice displayed improved survival, weight status and blood markers following 5-FU compared to control mice, indicating that UCN-01 can protect normal dividing tissues. The mechanism by which UCN-01 arrests normal cells in vivo was also examined. We have demonstrated that UCN-01 treatment in mice causes an increase in the G1 phase cell cycle proteins cdk4 and cyclin D, as well as the inhibitor p27. Phosphorylated Rb was also elevated in the arrested cells. These results are a departure from cell culture studies, in which inhibition of G1 phase cyclin dependent kinases led to hyposphosphorylation of Rb. Future investigation will be required to understand the mechanism of UCN-01 action. This is important information, especially for identification of alternate compounds which could provide the protection afforded by UCN-01.

Laser-induced transient grating analysis of dynamics of interaction between sensory rhodopsin II D75N and the HtrII transducer.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (DeltaHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K(1)-K(4)) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the DeltaHtrII portion was found to cause a DeltaHtrII-dependent increase in volume rising in 8 micros in the K(4) state and a drastic decrease in the diffusion coefficient (D) of K(4) relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of DeltaHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

Improving the accuracy of radiation pneumonitis dose response models

The prognosis for lung cancer patients remains poor. Five year survival rates have been reported to be 15%. Studies have shown that dose escalation to the tumor can lead to better local control and subsequently better overall survival. However, dose to lung tumor is limited by normal tissue toxicity. The most prevalent thoracic toxicity is radiation pneumonitis. In order to determine a safe dose that can be delivered to the healthy lung, researchers have turned to mathematical models predicting the rate of radiation pneumonitis. However, these models rely on simple metrics based on the dose-volume histogram and are not yet accurate enough to be used for dose escalation trials. The purpose of this work was to improve the fit of predictive risk models for radiation pneumonitis and to show the dosimetric benefit of using the models to guide patient treatment planning. The study was divided into 3 specific aims. The first two specifics aims were focused on improving the fit of the predictive model. In Specific Aim 1 we incorporated information about the spatial location of the lung dose distribution into a predictive model. In Specific Aim 2 we incorporated ventilation-based functional information into a predictive pneumonitis model. In the third specific aim a proof of principle virtual simulation was performed where a model-determined limit was used to scale the prescription dose. The data showed that for our patient cohort, the fit of the model to the data was not improved by incorporating spatial information. Although we were not able to achieve a significant improvement in model fit using pre-treatment ventilation, we show some promising results indicating that ventilation imaging can provide useful information about lung function in lung cancer patients. The virtual simulation trial demonstrated that using a personalized lung dose limit derived from a predictive model will result in a different prescription than what was achieved with the clinically used plan; thus demonstrating the utility of a normal tissue toxicity model in personalizing the prescription dose.

ANALYSIS OF THE CORYNEBACTERIUM PARVUM-INDUCED DECLINE OF MURINE NATURAL KILLER CELL-MEDIATED CYTOTOXICITY

Treatment of mice with the immunomodulating agent, Corynebacterium parvum (C. parvum), was shown to result in a severe and long-lasting depression of splenic natural killer (NK) cell-mediated cytotoxicity 5-21 days post-inoculation. Because NK cells have been implicated in immunosurveillance against malignancy (due to their spontaneous occurrence and rapid reactivity to a variety of histological types of tumors), as well as in resistance to established tumors, this decreased activity was of particular concern, since this effect is contrary to that which would be considered therapeutically desirable in cancer treatment (i.e. a potentiation of antitumor effector functions, including NK cell activity, would be expected to lead to a more effective destruction of malignant cells). Therefore, an analysis of the mechanism of this decline of splenic NK cell activity in C.parvum treated mice was undertaken.^ From in vitro co-culturing experiments, it was found that low NK-responsive C. parvum splenocytes were capable of reducing the normally high-reactivity of cells from untreated syngeneic mice to YAC-1 lymphoma, suggesting the presence of NK-directed suppressor cells in C. parvum treated animals. This was further supported by the demonstration of normal levels of cytotoxicity in C. parvum splenocyte preparations following Ficoll-Hypaque separation, which coincided with removal of the NK-suppressive capabilities of these cells. The T cell nature of these regulatory cells was indicated by (1) the failure of C. parvum to cause a reduction of NK cell activity, or the generation of NK-directed suppressor cells in T cell-deficient athymic mice, (2) the removal of C. parvum-induced suppression by T cell-depleting fractionation procedures or treatments, and (3) demonstration of suppression of NK cell activity by T cell-enriched C. parvum splenocytes. These studies suggest, therefore, that the eventual reduction of suppression by T cell elimination and/or inhibition, may result in a promotion of the antitumor effectiveness of C. parvum due to the contribution of "freed" NK effector cell activity.^ However, the temporary suppression of NK cell activity induced by C. parvum (reactivity of treated mice returns to normal levels within 28 days after C. parvum injection), may in fact be favorable in some situations, e.g. in bone marrow transplantation cases, since NK cells have been suggested to play a role also in the process of bone marrow graft rejection.^ Therefore, the discriminate use of agents such as C. parvum may allow for the controlled regulation of NK cell activity suggested to be necessary for the optimalization of therapeutic regimens. ^