Substantial and sustained reduction in under-5 mortality, diarrhea, and pneumonia in Oshikhandass, Pakistan: evidence from two longitudinal cohort studies 15 years apart.

BACKGROUND: Oshikhandass is a rural village in northern Pakistan where a 1989-1991 verbal autopsy study showed that diarrhea and pneumonia were the top causes of under-5 mortality. Intensive surveillance, active community health education and child health interventions were delivered in 1989-1996; here we assess improvements in under-5 mortality, diarrhea, and pneumonia over this period and 15 years later. METHODS: Two prospective open-cohort studies in Oshikhandass from 1989 to 1996 (Study 1) and 2011-2014 (Study 2) enrolled all children under age 60 months. Study staff trained using WHO guidelines, conducted weekly household surveillance and promoted knowledge on causes and management of diarrhea and pneumonia. Information about household characteristics and socioeconomic status was collected. Hurdle models were constructed to examine putative risk factors for diarrhea and pneumonia. RESULTS: Against a backdrop of considerable change in the socioeconomic status of the community, under-5 mortality, which declined over the course of Study 1 (from 114.3 to 79.5 deaths/1000 live births (LB) between 1989 and 1996), exceeded Sustainable Development Goal 3 by Study 2 (19.8 deaths/ 1000 LB). Reductions in diarrhea prevalence (20.3 to 2.2 days/ Child Year [CY]), incidence (2.1 to 0.5 episodes/ CY), and number of bloody diarrhea episodes (18.6 to 5.2%) seen during Study 1, were sustained in Study 2. Pneumonia incidence was 0.5 episodes /CY in Study 1 and 0.2/CY in Study 2; only 5% of episodes were categorized as severe or very severe in both studies. While no individual factors predicted a statistically significant difference in diarrhea or pneumonia episodes, the combined effect of water, toilet and housing materials was associated with a significant decrease in diarrhea; higher household income was the most protective factor for pneumonia in Study 1. CONCLUSIONS: We report a 4-fold decrease in overall childhood mortality, and a 2-fold decrease in childhood morbidity from diarrhea and pneumonia in a remote rural village in Pakistan between 1989 and 2014. We conclude that significant, sustainable improvements in child health may be achieved through improved socioeconomic status and promoting interactions between locally engaged health workers and the community, but that continued efforts are needed to improve health worker training, supervision, and the rational use of medications. TRIAL REGISTRATION: Not Applicable.

Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

Performance on a stage IV object-permanence task with standard and nonstandard covers.

Piaget's description of object concept development is based on success in performing a series of search tasks. However, failure to perform successfully may result from undeveloped aspects of the cognitive or motor system unrelated to a concept of objects. This study examined the role of perceptual-motor development in a typical Stage IV task by comparing performance given a standard cloth cover or a small card cover. Subjects were 10 infants 147--187 days old (median age = 160 days). A pass was defined as the retrieval of the toy within 30 sec on 2 out of 3 trials. The difference in performance between the 2 cover conditions was significant (p = .032). These results advise caution in interpreting Stage IV performance in terms of a developing object concept.

Cytosolic ADP-ribosylation factors are not required for endosome-endosome fusion but are necessary for GTP gamma S inhibition of fusion.

A specific role for ADP-ribosylation factors (ARFs) in in vitro endosome-endosome fusion has been proposed (Lenhard, J. M., Kahn, R. A., and Stahl, P. D. (1992) J. Biol. Chem. 267, 13047-13052). However, in vivo studies have failed to support a function for ARFs in the endocytic pathway, since an antagonist of ARF activities, brefeldin A, does not interfere with receptor internalization (Schonhorn, J. E., and Wessling-Resnick, M. (1994) Mol. Cell. Biochem. 135, 159-164). This controversy surrounding the exact function of ARF in endocytic vesicle traffic prompted us to critically re-examine the involvement of ARFs in cell-free endosome fusion. Cytosol depleted of ARF activity was capable of supporting in vitro endocytic vesicle fusion but failed to support inhibition of this reaction in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). Addition of purified ARF1 restored the ability of the ARF-depleted cytosol to inhibit endosome fusion when incubated with GTP gamma S. Both endocytic vesicle fusion and the GTP gamma S-mediated inhibition of vesicle fusion were unaffected by brefeldin A. Moreover, the ATP requirement and kinetics of cell-free fusion are not altered by brefeldin A or depletion of cytosolic ARFs. These results suggest that cytosolic ARFs are not necessary for endosomal vesicle fusion in vitro but are responsible for inhibition of fusion in the presence of GTP gamma S and cytosolic factors in a brefeldin A-resistant manner.

Studies on the inhibition of endosome fusion by GTPgammaS-bound ARF.

Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPgammaS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (beta COP) does not affect the capacity of GTPgammaS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPgammaS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPgammaS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.

Rab11 is associated with transferrin-containing recycling compartments in K562 cells.

3'RACE PCR was used to survey Rab transcripts synthesized by the human hematopoietic K562 cell line. Among the identified GTP-binding proteins, Rab11 was discovered. This result was unexpected since Rab11 previously had been found associated with polarized and secretory cells. Rab11 mRNA was abundant compared to that for other Rabs in K562 cells; protein levels represented 0.05-0.1% of total membrane protein. Localization of Rab11 using confocal immunofluoresence microscopy revealed extensive overlap with transferrin in recycling and/or exocytic compartments and suggests that Rab11 in non-polarized and non-secretory cells may play a role in the trafficking and recycling of internalized proteins.