Influenza A(H1N1)pdm09 infection in pregnant and non-pregnant women hospitalized in Singapore, May - December 2009.

OBJECTIVES: The aim of this study was to compare the demographic and clinical characteristics of pregnant women and non-pregnant women of childbearing age hospitalized with laboratory-confirmed influenza A(H1N1)pdm09 infection in Singapore, and to assess whether pregnancy was a risk factor associated with the development of influenza-related complications. STUDY DESIGN: Retrospective observational study. METHODS: We retrospectively identified and collected information from available medical records of all women admitted to three tertiary hospitals between 26 May 2009 and 31 December 2009 with laboratory-confirmed influenza A(H1N1)pdm09 infection who were either pregnant or non-pregnant and of childbearing age between 15 and 50 years. RESULTS: A total of 222 women, of whom 81 (36.5%) were pregnant, were hospitalized during the study period. Pregnant women were significantly more likely to be hospitalized with influenza A(H1N1)pdm09 infection than non-pregnant women of childbearing age (relative risk 26.3; 95% confidence interval: 20.1-34.6). Among those hospitalized, the proportion of pregnant women having at least one underlying medical condition that could predispose them to influenza-related complications was significantly lower than that of non-pregnant women (32.1% versus 56.0%, P < 0.001). The median time from onset of symptoms to administration of anti-viral drugs was significantly shorter among pregnant women than among non-pregnant women (three days versus five days, P < 0.001). The median length of stay in hospital was also significantly shorter among pregnant women than that of non-pregnant women (two days versus three days, P = 0.002). About 4.9% of pregnant women developed influenza-related complications, compared with 12.8% among non-pregnant women (P = 0.066). CONCLUSIONS: Pregnant women with influenza A(H1N1)pdm09 infection were at a higher risk of hospitalization. Upon hospitalization, they were not at a higher risk of developing influenza-related complications.

NANOG is a key factor for induction of pluripotency in bovine adult fibroblasts.

Since the first reports on isolation of pluripotent mouse embryonic stem (ES) cells 3 decades ago, there have been numerous attempts to derive ES cell lines from commercially important livestock species with limited success. The recent discovery that ectopic expression of a handful of stem cell-related genes was capable of inducing pluripotency in rodents and primates provided a novel approach to derivation of pluripotent stem cell lines. We used this approach in cattle and demonstrated that the ectopic expression of POU5F1 (also known as Oct4), SOX2, KLF4, and c-MYC alone was not sufficient for stable induction of pluripotency in bovine adult fibroblasts and that the additional expression of NANOG to the reprogramming cocktail was essential for the generation of stable bovine (b) induced pluripotent stem (iPS) cells. The resulting biPS cells were characterized by reverse-transcription PCR for a panel of ES marker genes. Immunocytochemical localization of POU5F1, SSEA-1, SSEA-4, and colorimetric alkaline phosphatase activity was measured in the iPS clones. The differentiation potential of the biPS cells was determined in vitro by expression of differentiation markers in embryoid bodies. Injection of biPS into immunocompromised mice resulted in teratomas containing cell types of the 3 germ lineages. This study reports the first generation of bovine induced pluripotent cell lines and paves the way for the use of biPS cells for biotechnological and agricultural purposes.

A novel, efficient method to derive bovine and mouse embryonic stem cells with in vivo differentiation potential by treatment with 5-azacytidine.

Pluripotent embryonic stem cells (ESCs) were first isolated nearly three decades ago from mice, yet efficient ESC isolation has been limited to rodents and primates to date. We report a novel and robust technique for isolating ESCs from mammalian pre-implantation embryos by altering the epigenotype of embryonic explants and using pressed zona pellucida-free blastocysts. We first examined this technique for murine ESC derivation. Compared with controls, murine ESCs were efficiently derived when explants were exposed to 1 µM 5-azacytidine, an epigenetic modifier that causes DNA demethylation (56.1% vs 31.6%; P < 0.01). Mouse ESCs stained positively for alkaline phosphatase, expressed markers of pluripotency including Oct4, Rex1 and SSEA1 and formed teratomas when injected into Severe Combined Immuno-Deficient (SCID) mice. The approach was subsequently used for bovine ESC derivation. In bovine a higher concentration of 5-azacytidine (5 µM) was required to elicit a response. This technique resulted in up to 18 times more efficient isolation of pluripotent cells than traditional methods (71.4% vs 4.0%; P < 0.001). These putative bovine ESCs expressed OCT4, REX1 mRNA and SSEA-1 and SSEA-4 proteins; and were able to form embryoid bodies in vitro and teratomas when injected in Severe Combined Immuno Deficient (SCID) mice. This is the first report on derivation of ESCs with both in vitro and in vivo differentiation potential in a livestock species.

Influenza A/H1N1 (2009) infection in pregnancy--an Asian perspective.

OBJECTIVE: To describe the characteristics of an obstetric population with influenza A/H1N1 (2009) infection, with a focus on the need for hospitalisation and complications. DESIGN: Cohort study. SETTING: Tertiary referral centre. POPULATION: Two hundred and eleven pregnant women with influenza A/H1N1 (2009) infection diagnosed by nasopharyngeal swab polymerase chain reaction (PCR). METHODS: Obstetric patients presenting to our centre were recruited and followed up. Data collected included demographic and clinical information. MAIN OUTCOME MEASURES: H1N1 and pregnancy complications, and hospitalisation needs. RESULTS: The median age of the cohort was 29.0 years (range 16-42 years), the median gestation at referral was 23.0 weeks (range 4-38 weeks), the median time interval between illness onset and presentation was 2.0 days (range 1-7 days), and the median time interval between illness onset and commencement of oseltamivir was 2.0 days (range 1-11 days). Hospital admission was significantly associated with the presence of co-morbidity (OR 4.14, 95% CI 1.82-9.37, P = 0.0001), breathlessness (OR 5.2, 95% CI 2.19-12.41, P = 0.0003) and sore throat (OR 0.35, 95% CI 0.16-0.73, P = 0.005). There were two cases of pneumonia complicating H1N1 infection, but no mortality. Nine cases developed pregnancy complications. All women recovered. CONCLUSIONS: The need for hospitalisation was significantly associated with breathlessness and co-morbidity. There was minimal morbidity and no mortality observed. We attribute this to early presentation, diagnosis and treatment.

Long-term efficacy and safety of fosamprenavir plus ritonavir versus lopinavir/ritonavir in combination with abacavir/lamivudine over 144 weeks.

PURPOSE: The KLEAN study extension assessed the long-term efficacy and safety of fosamprenavir-ritonavir (FPV/r) and lopinavir-ritonavir (LPV/r), both administered with abacavir/lamivudine (ABC/3TC) fixed dose combination, over 144 weeks. METHODS: KLEAN was an open-label, noninferiority study that randomised antiretroviral-naïve patients to FPV/r twice daily (bid) or LPV/r bid with ABC/3TC once daily (qd). Patients with a viral load of <400 copies/mL at Week 48 were eligible to participate in the KLEAN study extension (up to 144 weeks) and continued with their previously randomised therapy. RESULTS: The KLEAN study extension (48 to 144 weeks) randomized 199 patients. The proportion of TLOVR responders (HIV-1 RNA <50 copies/mL) at Week 144 was 73% and 60% in the FPV/r and LPV/r arms, respectively. The proportion of TLOVR responders (<50 copies/mL) was the same irrespective of baseline HIV-1 RNA (>100,000 or 100,000 copies/mL). The Week 144 median (interquartile range) change from baseline CD4+ cell count was 300 (236-433) cells/mm3 and 335 (225-444) cells/mm3 in the FPV/r and LPV/r arms, respectively. Diarrhea was the most frequently reported adverse event. A small proportion of patients (FPV/r, 13%; LPV/r, 9%) discontinued study medication due to adverse events. Three patients (FPV/r, 1; LPV/r, 2) experienced virological failure between Week 48 and Week 144. CONCLUSION: The findings of the KLEAN study extension (48 to 144 weeks) support durable viral suppression with both FPV/r and LPV/r treatment regimens when used in combination with ABC/3TC irrespective of viral load at baseline. Both regimens were well tolerated and had similar safety profiles.

DPC-083. DuPont Pharmaceuticals.

DPC-083 and DPC-961 are non-nucleoside reverse transcriptase inhibitors (NNRTIs) under development by DuPont Pharmaceuticals for the potential treatment of HIV infection [312865]. Phase I trials were completed by September 2000 [383661], and by April 2001, DPC-083 was in phase II trials as a once-daily oral dose in combination therapy, with phase III studies expected to begin in the third quarter of 2001 [392761]. NDA filing is anticipated for 2003, for which the company would expect an FDA expedited review [405548]. DPC-083 and DPC-961 possess antiviral activity against mutant variants of HIV-1, including the K103N mutant, and have the same potency as efavirenz against wild-type virus in rhesus monkeys [312865]. DuPont has also described a series of novel tricyclic quinazolinones which were found to possess noteworthy biological activity during investigation of DPC-083 and DPC-961.

The protonophore CCCP induces mitochondrial permeability transition without cytochrome c release in human osteosarcoma cells.

Mitochondrial permeability transition (MPT) and cytochrome c redistribution from mitochondria are two events associated with apoptosis. We investigated whether an MPT event obligatorily leads to cytochrome c release in vivo. We have previously shown that treatment of human osteosarcoma cells with the protonophore m-chlorophenylhydrazone (CCCP) for 6 h induces MPT and mitochondrial swelling without significant cell death. Here we demonstrate that release of cytochrome c does not occur and the cells remain viable even after 72 h of treatment with CCCP. Bax is not mobilized to mitochondria under these conditions. However, subsequent exposure of CCCP-treated cells to etoposide or staurosporine for 48 h results in rapid cell death and cytochrome c release that is accompanied by Bax association with mitochondria, demonstrating competency of these mitochondria to release cytochrome c with additional triggers. Our findings suggest that MPT is not a sufficient condition, in itself, to effect cytochrome c release.

Mitochondrial DNA deletions parallel age-linked decline in rat sensory nerve function.

In rats, the function of sensory nerves in the hind limb declines significantly with age. Normally aging rats and rats treated neonatally with capsaicin were studied here. Quantification of vascular response and substance P in young (3 months) and old (24 months) rats showed additive effects of age and capsaicin treatment. The levels in dorsal root ganglion of a particular deletion in mitochondrial DNA (mtDNA(4834)) were about 300-fold higher in old compared to young rats. Capsaicin treatment had no significant effect on mtDNA(4834) abundance. Dorsal root ganglia of old (but not young) rats were found to contain a spectrum of multiple deletions. The abundance of mtDNA(4834) in dorsal root ganglia from individual rats correlated strongly with their decline in vascular function, even where vascular responses were systematically depressed due to prior capsaicin treatment. One possibility is that mitochondrial DNA mutations directly lead to functional decline at mitochondrial and tissue levels. Alternatively, loss of mitochondrial DNA integrity and physiological decline may be consequences of the same factor, such as oxidative stress.

A perspective on tropical sprue.

A diagnosis of tropical sprue, an infrequent affliction of inhabitants and travelers in tropical regions, should be considered in patients with a compatible history, malabsorption, and chronic diarrhea. It can occur in either endemic or epidemic form and can be preceded by acute gastroenteritis. The cause of tropical sprue is still unknown, although most data support an infectious etiology. Therapeutic experience is greatest with folic acid and tetracycline. Most patients can be expected to recover with proper nutritional support, although relapses and slow responses occur.

Concurrent increase of oxidative DNA damage and lipid peroxidation together with mitochondrial DNA mutation in human lung tissues during aging--smoking enhances oxidative stress on the aged tissues.

Although mutation of mitochondrial DNA (mtDNA) in human tissues has been established to associate with intrinsic aging, the impact of environmental factors on the formation and accumulation of mtDNA mutations and oxidative DNA damage in human tissues is poorly understood. We have investigated the levels of mtDNA with the 4977-bp deletion and A3243G point mutation, oxidative DNA damage (indicated by the formation of 8-hydroxy-2'-deoxyguanosine, 8-OH-dG), and lipid peroxides in lung tissues from smokers and nonsmokers of subjects of different ages. The results showed concurrent age-dependent increase of the 4977-bp deleted mtDNA (P < 0.001), 8-OH-dG (P < 0.05), and lipid peroxides (P < 0.05) in the human lung. In the group of subjects above 60 years old, smokers had more extensive DNA damage and lipid peroxidation than did the nonsmokers. However, the levels of mtDNA with the 4977-bp deletion and A3243G point mutation in the lung of smokers were not significantly different from those of the age-matched nonsmokers. Taken together, these results suggest that accumulation of mtDNA with the 4977-bp deletion together with oxidative DNA damage and lipid peroxides is associated with aging and that smoking enhances oxidative damage in human lung tissues.

Estimate of perinatal mortality risk.

Perinatal mortality is usually calculated according to the World Health Organisation as stillbirth and first week mortality at a specified week of gestation divided by all births at that same gestational week. This is not a meaningful indicator of the risk of future perinatal death for a living fetus. We have developed an approach to estimate the prospective risk of perinatal mortality. Data were derived from the Perinatal Database of the Netherlands. We calculated the prospective risk of perinatal mortality by dividing all future perinatal deaths from a certain week of gestation by all fetuses that remained undelivered. Using this statistic there is a decline in risk from 16 to 39 gestational weeks and an increase from 39 weeks onwards.

Beta-thalassemia mutations in Indonesia and their linkage to beta haplotypes.

A total of 72 chromosomes from 36 Indonesian patients, 23 with beta-thalassemia major and 13 with Hb E-beta-thalassemia, were analyzed by specific oligonucleotide hybridization after DNA amplification. Thirteen had the beta E mutation (codon 26 GAG----AAG). Of the 59-beta-thalassemic chromosomes, 32 were of the variant IVS-1 nt5 (G----C). Seven had the mutation IVS-2 nt654 (C----T), one had the mutation codon 41/42 (deletion CTTT), and one had the mutation codon 17 (AAG----TAG). Another six with the mutation IVS-1 nt1 (G----T), one with the mutation IVS-1 nt1 (G----A), four with the mutation codon 15 (TGG----TAG), one with a mutation codon 30 (AGG----ACG), and one with a mutation codon 35 (deletion C) were first identified by direct sequencing of a patient's genomic DNA followed by further hybridizing other patients' DNA with the appropriate oligonucleotide probes. Five did not carry the common mutations previously described in Asian populations. The four most prevalent mutations encountered made up 83% of the total number of beta-thalassemic chromosomes studied. The most common mutation, IVS-1 nt5 (G----C), was mostly associated with two different haplotypes.

Nipple aspirate fluids in adult nonlactating women--lactose content, cationic Na+, K+, Na+/K+ ratio, and coloration.

The presence of lactose in nipple secretions is considered biochemical evidence of breast secretory activity, and has been reported to occur more frequently in white compared to brownish or green colored breast fluid. We studied lactose, Na+, and K+ concentrations, the Na+/K+ ratio, and the coloration of nipple aspirate fluid (NAF) from 49 nonpregnant women. A significant relationship was found between the concentrations of lactose, Na+, and K+, and age and the coloration of NAF. Lactose was present in 22/49 (44.8%) of the NAF samples and declined with age from 100% positivity in women less than or equal to 29 years to 29% in those less than or equal to 35 years. In NAF of deep yellow, brown and green colorations, only traces of lactose were found. Na+ and K+ increased with age and with darker colorations compared to white, pale yellow, or colorless NAF. Lactose was present in NAF samples from both parous and nulliparous younger women, indicating that the breasts of many nonpregnant women respond to prolactin stimulation; hence, lactose may provide a simple marker indicating active physiologic secretory activity of the breast. As reported previously, NAF of darker coloration, containing elevated levels of cholesterol, cholesterol oxidation products, and other substances, suggests retention and impaired reabsorption of these and other products of secretion. Because of the secretion and temporary retention by the breast glands of chemical substances of exogenous and endogenous origin, including mutagens and carcinogens, lactose concentration and coloration of NAF may be useful as markers of secretion and reabsorption in future physiologically based clinical and epidemiologic studies of the pathogenesis of breast disease.

Quantitation of hepatitis B virus DNA in serum of patients with hepatoma.

Hepatitis B virus (HBV) DNA in the serum of 31 patients with histologically confirmed primary hepatocellular carcinoma (PHC) from Malaysia and Indonesia was quantitated by densitometric scanning of autoradiograms obtained by Southern blot DNA hybridization, after electrophoresis using a 32P DNA cloned into plasmid pBR325 as a probe. This quantitation after electrophoresis is more informative than the usual spot hybridization technique. Five of the 31 sera were positive for HBV DNA. Levels ranged between 1.36 pq and 143.18 pq per ml of serum, and the levels of HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe in the serum were serologically determined. All five sera positive for HBV DNA were also positive for HBsAg. Three of the five positive for HBV DNA were positive for HBeAg and negative for anti-HBe. Two of the sera positive for HBV DNA were negative for HBeAg but positive for anti-HBe. All sera negative for HBV DNA were also negative for HBeAg. Many sera which were negative for HBV DNA and HBeAg were positive for HBsAg. Of the 31 sera from PHC patients, 23 had at least one HBV marker positive (74.2%).

Elevated G gamma gene expression with specific beta S gene haplotype, normal gamma gene maps and presence of the Xmn I site -158 5' to the G gamma gene in Indian sickle cell anemia.

In nine Indian patients ranging in age between four and 61 years, with mild Hb SS disease and very high Hb F levels, the G gamma globin chain levels of their fetal hemoglobin ranged between 64.0% and 70.0%, with a mean of 68.1% (S.D. +/- 2.6) of the total amount of gamma-globin chains. Eight of the nine patients were homozygous for a specific beta S gene haplotype #31. The other one was doubly heterozygous for the same specific haplotype and another haplotype, which differed from haplotype #31 by the presence of Bam HI site 3' to the beta gene and absence of Pvu II site 5' to the psi beta gene. The gamma gene organization studied by Pst I restriction enzyme analysis was found to be normal and the Xmn I site -158 5' to G gamma gene was present in all patients examined.

Sickle cell anemia associated with alpha-thalassemia in Malaysian Indians.

The Indian rubber estate workers in Negri Sembilan, Malaysia, who originated from Orissa in India were found to have a high frequency of Hb S (Joishy SK, Hassan K: Clin Res 28:280, 1980). Unlike the usually severe clinical picture of sickle cell anemia seen in African and American blacks, the clinical picture of the disease in this population was mild and many have reached old age. We studied the leukocyte DNA of 12 patients with sickle cell anemia, ranging in age from 4 to 61 years and 30 sickle cell trait carriers, ranging in age from 7 to 63 years, for the presence of alpha-globin gene deletions by gene mapping according to Southern (Southern EM: J Mol Biol 98:503, 1975), using alpha- and zeta-globin gene probes obtained by nick translation of the alpha- and zeta-globin genes cloned into plasmid. All 12 sickle cell anemia patients were found to have alpha-thalassemia2 (alpha-thal2), either in the homozygous or heterozygous condition. Of the Hb S trait carriers, six did not have alpha-thal2 or alpha-thal1 and 24 had alpha-thal2 (15 heterozygous, 9 homozygous). Seven of these Hb S trait carriers with alpha-thal2 had an additional gene abnormality. Five of them had a fast-moving Eco RI fragment 5.6 kb long that hybridized with zeta-specific probe but not with alpha-specific probe. An unusual DNA pattern of a different type was further found in the other two. Bgl II restriction analysis showed that the alpha-thal2 was mostly of the rightward deletion alpha-thal1 genotype. None of the sickle cell anemia patients and Hb S trait carriers had deletion type alpha-thal1. The sickle cell anemia patients had very high levels of Hb F and low levels of Hb A2. The Hb S trait carriers with alpha-thal2 had relatively low levels of Hb S.

Depression after stroke: a hospital treatment survey.

Treatment with anti-depressant drugs of 61 inpatients admitted on account of stroke was studied. Eighteen patients (30%) were being so treated. Only 5 patients (17%) with dysphasia were being treated with anti-depressant drugs compared with 13 patients (41%) without dysphasia (P less than 0.05). Dosage levels used were generally low and only one patient had been referred for a psychiatric opinion. We suggest that the difference in anti-depressant usage is because dysphasic patients are more difficult to assess and therefore less likely to have depressive symptoms recognized and treatment given. We also think that psychiatric referral should be used more often for assessment of stroke patients and for advice about treatment.

The influence of warfarin of levamisole on the incidence of metastases following local irradiation of a solid tumor.

KHT sarcomas were implanted in the right rear legs of C3H mice. An x-ray dose of 6000 rad, delivered in ten equal fractions over 12 days, resulted in 60% local tumor control, but 83% of these mice developed metastases. Three strategies to use the tumoricidal effect of x-radiation and reduce the incidence of metastases were compared. A modification of the fractionation scheme to deliver an initial large fraction of 1800 rad followed by seven 600-rad fractions resulted in a decreased incidence of metastases compared with the same dose delivered in ten equal fractions. The use of warfarin anticoagulation during the ten-fraction course of radiation resulted in a small decrease in the incidence of metastases. Immune stimulation with levamisole, injected subcutaneously every second day during the irradiation, also resulted in a decrease in the incidence of metastases. However, when warfarin or levamisole were combined with the eight-fraction radiation scheme there were fewer metastases than following the ten-fraction scheme. The combination of the eight-fraction radiation course with levamisole also produced a significant increase in primary tumor control. In this treatment regimen, therefore, levamisole appears to act as a radiation sensitizer. An hypothesis to explain this action is proposed.

Dose local x-irradiation of a tumor increase the incidence of metastases?

KHT sarcomas were implanted into the right rear legs of C3H mice. Animals receiving no treatment died at 32 days with a 67% incidence of metastatic tumors. A dose of 6000 rads in ten fractions over 12 days, starting ten days after implant, delivered to the primary tumor (with the remainder of the body shielded) produced 55% local tumor control. However, 83% of the mice with local tumor control, subsequently developed metastatic lesions. Amputation of the tumor-bearing leg either prior to the start of radiation treatment, at the end of treatment or at intervals during treatment or sham treatment, indicated that 1) metastases occurred predominantly during the initial three radiation fractions and 2) the increased incidence of metastases following irradiation was a result of the manipulations associated with the irradiation and not the irradiation per se. Irradiation did not change the distribution of organ sites involved with metastatic tumors compared to untreated or sham-treated tumors.