Shigella as a Cause of Diarrhea Hospitalization in Children Under Five: Evaluation by Conventional and Molecular Methods.

BACKGROUND AND OBJECTIVES: Shigella is an important cause of diarrhea in children under five, often missed by conventional laboratory methods. Blood in stools has always been a syndromic indicator for Shigella diarrhea, but most cases present with watery diarrhea without blood. This study aimed to determine the frequency of Shigella detected by molecular and conventional methods in children under five. Additionally, we aimed to study the clinical profile and outcome of children with Shigella diarrhea managed as per current diarrhea treatment guidelines. METHODS: In this hospital-based prospective observational study, stool samples from 150 children (age range: one month to five years) with acute diarrhea (duration < seven days) were subjected to routine microscopic examination, stool culture, and DNA extraction. The extracted DNA from stored stool samples was subjected to polymerase chain reaction (PCR) amplification using a specific primer for the invasion plasmid antigen H gene sequence (ipaH) gene at 424 bp. Results were interpreted in the context of the percentage of isolation of Shigella by molecular (PCR) and conventional methods (stool microscopy and culture) and the follow-up outcome in terms of recurrence of diarrhea or dysentery and growth faltering over three months after discharge. RESULTS: Shigella infection was diagnosed in stool samples by PCR from 13 (8.7%) children, whereas it was isolated by conventional stool culture in only one (0.7%) child. The sensitivity of culture was only 7.7% against PCR for the diagnosis of Shigella infection, whereas blood in stools had a sensitivity of 15.4%. The majority of Shigella PCR-positive cases (11 out of 13) presented with non-bloody diarrhea. None of the evaluated clinical predictors had a significant association with the Shigella infection. No statistically significant difference was found between PCR-positive and PCR-negative children at the end of follow-up (P>0.05). CONCLUSION: The majority of children with Shigella infection present with watery diarrhea rather than bloody diarrhea, and a history of blood in stools is a poor marker for the diagnosis of shigellosis. The diagnostic performance of stool culture is also very low compared to stool PCR for the diagnosis of Shigella diarrhea.

A randomised trial to compare 200 mg micronised progesterone effervescent vaginal tablet daily with 250 mg intramuscular 17 alpha hydroxy progesterone caproate weekly for prevention of recurrent preterm birth.

For prevention of a recurrent preterm birth (PTB), intramuscular 17-α-hydroxy progesterone caproate (IM 17 OHPC) weekly is recommended. Vaginal progesterone is preferred for women at risk for PTB due to a short cervical length, but may be useful in women with a prior PTB. However, there is no consensus about the optimal vaginal formulation or its efficacy as compared to 17 OHPC to prevent recurrent PTB. We randomised 100 women with a singleton pregnancy between 16 and 24 weeks of gestation and ≥ one prior spontaneous PTB, of a singleton (>16 to <37 weeks of gestation) to receive the 200 mg vaginal progesterone effervescent tablet daily (Group A) or IM 17-OHPC, 250 mg weekly (Group B) till 37 weeks of gestation or delivery. The spontaneous PTB rate of <37 weeks was similar (20% in Group A and 20.8% in Group B, p = .918). The PTB rate of <34 weeks or <28 weeks were also comparable. The mean birth weight and other neonatal outcomes were similar in the two groups. Two neonates in Group A and four neonates in Group B required NICU admission, one of whom (Group B) died due to prematurity. Twenty percent of women in Group A and 29.2% in Group B reported adverse effects from their respective study medications (p = .408, NS). Thus, there did not appear to be a difference between vaginal progesterone and 17-OHPC when used for the prevention of a recurrent PTB. Impact statement What is already known on this subject? Progesterone administration is useful for prevention of a recurrent preterm birth (PTB) and these women are prescribed the intramuscular 17-α-hydroxy progesterone caproate (IM 17 OHPC), 250 mg, weekly. Some studies found that vaginal progesterone (once daily) is also beneficial in these women, but there is no consensus regarding its efficacy when compared to 17 OHPC, or its optimal formulation and dose. What do the results of this study add? In the present study, 100 women with a singleton pregnancy between 16 and 24 weeks of gestation and ≥ one prior spontaneous singleton PTB or mid-trimester abortion were randomised to receive 200 mg of vaginal progesterone effervescent tablet daily (Group A) or 250 mg IM 17-OHPC weekly (Group B) till 37 weeks of gestation or delivery. The spontaneous PTB rate <37 weeks was similar in the two groups (20% in Group A and 20.8% in Group B, p = .918). The PTB rate <34 weeks or <28 weeks were also comparable. The mean birth weight and other neonatal outcomes were similar. Twenty percent of women in Group A and 29.2% of women in Group B reported adverse effects from their respective study medications (p = .408, NS). Thus, there did not appear to be a difference between the vaginal progesterone effervescent tablet and 17-OHPC when used for the prevention of a recurrent PTB. What are the implications of these findings for clinical practice and/or further research? The vaginal progesterone effervescent tablet may be a suitable alternative to IM 17 OHPC to prevent recurrent PTB. Future studies should identify the most appropriate route (IM or vaginal) and vaginal progesterone formulation for PTB prevention in women at risk for a recurrent PTB and in women with a short cervical length.

Cell division cycle 25B phosphatase is essential for benzo(a)pyrene-7,8-Diol-9,10-epoxide induced neoplastic transformation.

Cell division cycle 25B (Cdc25B) phosphatase controls entry into mitosis and regulates recovery from G2-M checkpoint-induced arrest. In the present study, we show that exposure of diploid mouse embryonic fibroblasts (MEF) to the ultimate carcinogen anti-benzo(a)pyrene (BP)-7,8-diol-9,10-epoxide (anti-BPDE) resulted in a concentration- and time-dependent increase in Cdc25B protein levels. Chronic exposure of wild-type (Cdc25B+/+) MEFs to anti-BPDE (0.1 micromol/L) caused neoplastic transformation characterized by colony formation in culture and tumor production in nude mice. In contrast, the Cdc25B null MEFs were resistant to anti-BPDE-induced transformation. Furthermore, a carcinogenic dose of the parent hydrocarbon (BP) increased Cdc25B protein levels in the target organ, lung. The biological importance of elevated Cdc25B levels was documented by the early reentry into mitosis of cells overexpressing ectopic Cdc25B levels even in the presence of DNA damage following anti-BPDE exposure, whereas control cells resumed only after DNA damage was repaired. We conclude that Cdc25B has an essential role in anti-BPDE-induced neoplastic transformation, including regulation of cell cycle resumption in the presence of DNA damage.

Induction of Cdc25B regulates cell cycle resumption after genotoxic stress.

Cdc25 phosphatases propel cell cycle progression by activating cyclin-dependent kinases (Cdk). DNA damage is generally thought to inhibit Cdc25 functionality by inducing proteasomal degradation of Cdc25A and phosphorylation-mediated sequestration of Cdc25B and Cdc25C to the cytoplasm. More recently, a critical role for Cdc25B in the resumption of cell cycle progression through mitosis after DNA damage has been identified. In this study, the fate of Cdc25B after mechanistically distinct DNA-damaging agents (etoposide, cisplatin, bleomycin, ionizing irradiation, or UV irradiation) was examined, and surprisingly a rapid increase in cellular Cdc25B levels was observed after DNA damage. Using UV irradiation as the prototypic damaging agent, we found that the increase in Cdc25B levels was checkpoint dependent and was controlled by a p53-independent mechanism. Cdc25B levels controlled the number of cells progressing into mitosis after UV, but they did not affect G(2)-M checkpoint engagement immediately after DNA damage. Increased Cdc25B reduced the time required for cell cycle resumption. These data support a model in which Cdc25B accumulation is an important anticipatory event for cell cycle resumption after DNA damage.

Airing out an antioxidant role for the tumor suppressor p53.

The tumor suppressor p53 exerts its activity by preventing DNA-damaged cells from dividing until either the chromosomal repair is effected or the cell undergoes apoptosis. Reactive oxygen species (ROS) are enhanced through the action of p53-mediated transcription of apoptosis-promoting genes; however, p53 also can promote the expression of many antioxidant genes that prevent apoptosis. New research indicates that in low cellular stress, low concentrations of p53 induce the expression of antioxidant genes, whereas in severe cellular stress, high concentrations of p53 promote the expression of genes that contribute to ROS formation and p53-mediated apoptosis. p53-depleted cells injected into athymic mice increased significantly in tumor volume, whereas injected p53(+/+) cells did not grow to the same degree. Interestingly, administration of the antioxidant compound N-acetylcysteine (NAC) inhibited the growth of tumor volume in p53-depleted injected cells, and NAC supplementation of p53(-/-) mice from birth greatly decreased the number of karyotype abnormalities and tumors formed in these mice by six months of age. Thus, under normal (or low stress) conditions, p53 appears to have an antioxidant role that protects cells from oxidative DNA damage and although this effect might depend on the concentration of p53, other cellular factors likely participate in a cell's final fate.

Redox regulation of Cdc25B by cell-active quinolinediones.

Intracellular reduction and oxidation pathways regulate protein functionality through both reversible and irreversible mechanisms. The Cdc25 phosphatases, which control cell cycle progression, are potential subjects of oxidative regulation. Many of the more potent Cdc25 phosphatase inhibitors reported to date are quinones, which are capable of redox cycling. Therefore, we used the previously characterized quinolinedione Cdc25 inhibitor DA3003-1 [NSC 663284 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] and a newly synthesized congener JUN1111 [7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione] to test the hypothesis that quinone inhibitors of Cdc25 regulate phosphatase activity through redox mechanisms. Like DA3003-1, JUN1111 selectively inhibited Cdc25 phosphatases in vitro in an irreversible, time-dependent manner and arrested cells in the G1 and G2/M phases of the cell cycle. It is noteworthy that both DA3003-1 and JUN1111 directly inhibited Cdc25B activity in cells. Depletion of glutathione increased cellular sensitivity to DA3003-1 and JUN1111, and in vitro Cdc25B inhibition by these compounds was sensitive to pH, catalase, and reductants (dithiothreitol and glutathione), consistent with oxidative inactivation. In addition, both DA3003-1 and JUN1111 rapidly generated intracellular reactive oxygen species. Analysis of Cdc25B by mass spectrometry revealed sulfonic acid formation on the catalytic cysteine of Cdc25B after in vitro treatment with DA3003-1. These results indicate that irreversible oxidation of the catalytic cysteine of Cdc25B is indeed a mechanism by which these quinolinediones inactivate this protein phosphatase.

Induction of Cdc25B expression by epidermal growth factor and transforming growth factor-alpha.

The dual specificity protein phosphatase Cdc25B regulates of the mitotic cell cycle checkpoint and is over expressed in human tumors. Given the importance of growth factors in initiating and sustaining cell proliferation, we examined their effects on Cdc25B protein expression in human cancer cells. Within 1h after epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) treatment, Cdc25B protein levels increased in growth factor responsive A549 and SCC25 cells, but not in non-responsive MDA-MB-231 cells. A functional consequence of elevated Cdc25B was implied by the concomitant decrease in phosphorylated cyclin dependent kinase, a known Cdc25B substrate, after growth factor treatment of A549 and SCC25 cells. The EGF-mediated induction of Cdc25B required a functional EGF receptor (ErbB1), as mouse embryonic fibroblasts lacking ErbB1 did not have increased Cdc25B levels after EGF treatment. Moreover, the EGFR receptor-selective tyrosine kinase inhibitor AG1478 and mitogen activated kinase kinase inhibitor U0126 blocked growth factor-mediated Cdc25B induction. Thus, EGF and TGF-alpha appear to induce cellular Cdc25B through the mitogen-activated protein kinase pathway.