Surveillance of antimicrobial resistance of Streptococcus pneumoniae and Haemophilus influenzae in children with pneumonia

In order to identify optimal therapy for children with bacterial pneumonia, Pakistan's ARI Program, in collaboration with the National Institute of Health (NIH), Islamabad, undertook a national surveillance of antimicrobial resistance in S. pneumoniae and H. influenzae. The project was carried out at selected urban and peripheral sites in 6 different regions of Pakistan, in 1991–92. Nasopharyngeal (NP) specimens and blood cultures were obtained from children with pneumonia diagnosed in the outpatient clinic of participating facilities. Organisms were isolated by local hospital laboratories and sent to NIH for confirmation, serotyping and antimicrobial susceptibility testing. Following were the aims of the study (i) to determine the antimicrobial resistance patterns of S. pneumoniae and H. influenzae in children aged 2–59 months; (ii) to determine the ability of selected laboratories to identify and effectively transport isolates of S. pneumoniae and H. influenzae cultured from nasopharyngeal and blood specimens; (iii) to validate the comparability of resistance patterns for nasopharyngeal and blood isolates of S. pneumoniae and H. influenzae from children with pneumonia; and (iv) to examine the effect of drug resistance and laboratory error on the cost of effectively treating children with ARI. ^ A total of 1293 children with ARI were included in the study: 969 (75%) from urban areas and 324 (25%) from rural parts of the country. Of 1293, there were 786 (61%) male and 507 (39%) female children. The resistance rate of S. pneumoniae to various antibiotics among the urban children with ARI was: TMP/SMX (62%); chloramphenicol (23%); penicillin (5%); tetracycline (16%); and ampicillin/amoxicillin (0%). The rates of resistance of H. influenzae were higher than S. pneumoniae: TMP/SMX (85%); chloramphenicol (62%); penicillin (59%); ampicillin/amoxicillin (46%); and tetracycline (100%). There were similar rates of resistance to each antimicrobial agent among isolates from the rural children. ^ Of a total 614 specimens that were tested for antimicrobial susceptibility, 432 (70.4%) were resistant to TMP/SMX and 93 (15.2%) were resistant to antimicrobial agents other than TMP/SMX viz. ampicillin/amoxicillin, chloramphenicol, penicillin, and tetracycline. ^ The sensitivity and positive predictive value of peripheral laboratories for H. influenzae were 99% and 65%, respectively. Similarly, the sensitivity and positive predictive value of peripheral laboratory tests compared to gold standard i.e. NIH laboratory, for S. pneumoniae were 99% and 54%, respectively. ^ The sensitivity and positive predictive value of nasopharyngeal specimens compared to blood cultures (gold standard), isolated by the peripheral laboratories, for H. influenzae were 88% and 11%, and for S. pneumoniae 92% and 39%, respectively. (Abstract shortened by UMI.)^

Non B-DNA structures and genomic instability associated with myotonic dystrophy type 2 (CCTG).(CAGG) repeats

There are many diseases associated with the expansion of DNA repeats in humans. Myotonic dystrophy type 2 is one of such diseases, characterized by expansions of a (CCTG)•(CAGG) repeat tract in intron 1 of zinc finger protein 9 (ZNF9) in chromosome 3q21.3. The DM2 repeat tract contains a flanking region 5' to the tract that consists of a polymorphic repetitive sequence (TG)14-25(TCTG)4-11(CCTG) n. The (CCTG)•(CAGG) repeat is typically 11-26 repeats in persons without the disease, but can expand up to 11,000 repeats in affected individuals, which is the largest expansion seen in DNA repeat diseases to date. This DNA tract remains one of the least characterized disease-associated DNA repeats, and mechanisms causing the repeat expansion in humans have yet to be elucidated. Alternative, non B-DNA structures formed by the expanded repeats are typical in DNA repeat expansion diseases. These sequences may promote instability of the repeat tracts. I determined that slipped strand structure formation occurs for (CCTG)•(CAGG) repeats at a length of 42 or more. In addition, Z-DNA structure forms in the flanking human sequence adjacent to the (CCTG)•(CAGG) repeat tract. I have also performed genetic assays in E. coli cells and results indicate that the (CCTG)•(CAGG) repeats are more similar to the highly unstable (CTG)•(CAG) repeat tracts seen in Huntington's disease and myotonic dystrophy type 1, than to those of the more stable (ATTCT)•(AGAAT) repeat tracts of spinocerebellar ataxia type 10. This instability, however, is RecA-independent in the (CCTG)•(CAGG) and (ATTCT)•(AGAAT) repeats, whereas the instability is RecA-dependent in the (CTG)•(CAG) repeats. Structural studies of the (CCTG)•(CAGG) repeat tract and the flanking sequence, as well as genetic selection assays may reveal the mechanisms responsible for the repeat instability in E. coli, and this may lead to a better understanding of the mechanisms contributing to the human disease state. ^

Associations between vaccination and childhood cancers in Texas counties

Background. In Dr. Mel Greaves "delayed-infection hypothesis," postponed exposure to common infections increases the likelihood of childhood cancer. Hygienic advancements in developed countries have reduced children's exposure to pathogens and children encounter common infectious agents at an older age with an immune system unable to deal with the foreign antigens. Vaccinations may be considered to be simulated infections as they prompt an antigenic response by the immune system. Vaccinations may regulate the risk of childhood cancer by modulating the immune system. The aim of the study was to determine if children born in Texas counties with higher levels of vaccination coverage were at a reduced risk for childhood cancer.^ Methods. We conducted a case-control study to examine the risk of childhood cancers, specifically leukemia, brain tumors, and non-Hodgkin lymphoma, in relation to vaccination rates in Texas counties. We utilized a multilevel mixed-effects regression model of the individual data from the Texas Cancer Registry (TCR) with group-level exposure data (i.e., the county- and public health region-level vaccination rates).^ Results. Utilizing county-level vaccination rates and controlling for child's sex, birth year, ethnicity, birth weight, and mother's age at child's birth the hepatitis B vaccine revealed negative associations with developing all cancer types (OR = 0.81, 95% CI: 0.67–0.98) and acute lymphoblastic leukemia (ALL) (OR = 0.63, 95% CI: 0.46–0.88). The decreased risk for ALL was also evident for the inactivated polio vaccine (IPV) (OR = 0.67, 95% CI: 0.49–0.92) and 4-3-1-3-3 vaccination series (OR = 0.62, 95% CI: 0.44-0.87). Using public health region vaccine coverage levels, an inverse association between the Haemophilus influenzae type b (Hib) vaccine and ALL (OR: 0.58; 95% CI: 0.42–0.82) was present. Conversely, the measles, mumps, and rubella (MMR) vaccine resulted in a positive association with developing non-Hodgkin lymphoma (OR = 2.81, 95% CI: 1.27–6.22). ^

DOUBLE-STRAND BREAK REPAIR PATHWAYS IN DNA STRUCTURE-INDUCED GENETIC INSTABILITY

Genetic instability in mammalian cells can occur by many different mechanisms. In the absence of exogenous sources of DNA damage, the DNA structure itself has been implicated in genetic instability. When the canonical B-DNA helix is naturally altered to form a non-canonical DNA structure such as a Z-DNA or H-DNA, this can lead to genetic instability in the form of DNA double-strand breaks (DSBs) (1, 2). Our laboratory found that the stability of these non-B DNA structures was different in mammals versus Escherichia coli (E.coli) bacteria (1, 2). One explanation for the difference between these species may be a result of how DSBs are repaired within each species. Non-homologous end-joining (NHEJ) is primed to repair DSBs in mammalian cells, while bacteria that lack NHEJ (such as E.coli), utilize homologous recombination (HR) to repair DSBs. To investigate the role of the error-prone NHEJ repair pathway in DNA structure-induced genetic instability, E.coli cells were modified to express genes to allow for a functional NHEJ system under different HR backgrounds. The Mycobacterium tuberculosis NHEJ sufficient system is composed of Ku and Ligase D (LigD) (3). These inducible NHEJ components were expressed individually and together in E.coli cells, with or without functional HR (RecA/RecB), and the Z-DNA and H-DNA-induced mutations were characterized. The Z-DNA structure gave rise to higher mutation frequencies compared to the controls, regardless of the DSB repair pathway(s) available; however, the type of mutants produced after repair was greatly dictated on the available DSB repair system, indicated by the shift from 2% large-scale deletions in the total mutant population to 24% large-scale deletions when NHEJ was present (4). This suggests that NHEJ has a role in the large deletions induced by Z-DNA-forming sequences. H-DNA structure, however, did not exhibit an increase in mutagenesis in the newly engineered E.coli environment, suggesting the involvement of other factors in regulating H-DNA formation/stability in bacterial cells. Accurate repair by established DNA DSB repair pathways is essential to maintain the stability of eukaryotic and prokaryotic genomes and our results suggest that an error-prone NHEJ pathway was involved in non-B DNA structure-induced mutagenesis in both prokaryotes and eukaryotes.

The in vivo and in vitro formation of sticky DNA and the GAA.TTC trinucleotide repeat associated with Friedreich's ataxia

Friedreich's ataxia is caused by the expansion of the GAA•TTC trinucleotide repeat sequence located in intron 1 of the frataxin gene. The long GAA•TTC repeats are known to form several non-B DNA structures including hairpins, triplexes, parallel DNA and sticky DNA. Therefore it is believed that alternative DNA structures play a role in the loss of mRNA transcript and functional frataxin protein in FRDA patients. We wanted to further elucidate the characteristics for formation and stability of sticky DNA by evaluating the structure in a plasmid based system in vitro and in vivo in Escherichia coli. The negative supercoil density of plasmids harboring different lengths of GAA•TTC repeats, as well as either one or two repeat tracts were studied in E. coli to determine if plasmids containing two long tracts (≥60 repeats) in a direct repeat orientation would have a different topological effect in vivo compared to plasmids that harbored only one GAA•TTC tract or two tracts of < 60 repeats. The experiments revealed that, in fact, sticky DNA forming plasmids had a lower average negative supercoil density (-σ) compared to all other control plasmids used that had the potential to form other non-B DNA structures such as triplexes or Z-DNA. Also, the requirements for in vitro dissociation and reconstitution of the DNA•DNA associated region of sticky DNA were evaluated. Results conclude that the two repeat tracts associate in the presence of negative supercoiling and MgCl 2 or MnCl2 in a time and concentration-dependent manner. Interaction of the repeat sequences was not observed in the absence of negative supercoiling and/or MgCl2 or in the presence of other monovalent or divalent cations, indicating that supercoiling and quite specific cations are needed for the association of sticky DNA. These are the first experiments studying a more specific role of supercoiling and cation influence on this DNA conformation. To support our model of the topological effects of sticky DNA in plasmids, changes in sticky DNA band migration was measured with reference to the linear DNA after treatment with increasing concentrations of ethidium bromide (EtBr). The presence of independent negative supercoil domains was confirmed by this method and found to be segregated by the DNA-DNA associated region. Sequence-specific polyamide molecules were used to test the effect of binding of the ligands to the GAA•TTC repeats on the inhibition of sticky DNA. The destabilization of the sticky DNA conformation in vitro through this binding of the polyamides demonstrated the first conceptual therapeutic approach for the treatment of FRDA at the DNA molecular level. ^ Thus, examining the properties of sticky DNA formed by these long repeat tracts is important in the elucidation of the possible role of sticky DNA in Friedreich's ataxia. ^

Survey of VFC provider knowledge of catch-up regimens and contraindications to vaccination in Houston, Texas

In 2004, Houston had one of the lowest childhood immunization levels among major metropolitan cities in the United States at 65% for the 4:3:1:3:3 vaccination series. Delays in the receipt of scheduled vaccinations may be related to missed opportunities due to health care provider lack of knowledge about catch-up regimens and contraindications for pediatric vaccination. The objectives of this study are to identify, measure, and report on VFC provider-practice characteristics, knowledge of catch-up regimens and contraindications, and use of Reminder recall (R/R) and moved or gone elsewhere (MOGE) practices among providers with high (>80%) and low (<70%) immunization coverage among 19-35 month old children. The sampling frame consists of 187 Vaccines for Children (VFC) providers with 2004 clinic assessment software application (CASA) scores. Data were collected by personal interview with each participating practice provider. Only ten VFC providers were successful at maximizing vaccinations for every vignette and no provider administered the maximum possible number of vaccinations at visit 2 for all six vignettes. Both coverage groups administered polio conjugate vaccine (PCV), haemophilus influenza type b (Hib), and diphtheria, tetanus and acellular pertussis (DTaP) most frequently and omitted most frequently varicella zoster vaccine (VZV) and measles, mumps, and rubella (MMR) vaccine. ^

Mechanisms of liposomal all-{\it trans\/} retinoic acid and vitamin D3 induced inhibition of cell proliferation and stimulation of apoptosis in aggressive B-cell non-Hodgkin's lymphoma

The Non-Hodgkin's Lymphoma (NHLs) are neoplasms of the immune system. Currently, less than 1% of the etiology of the 22,000 newly diagnosed lymphoma cases in the U.S.A. every year is known. This disease has a significant prevalence and high mortality rate. Cell growth in lymphomas has been shown to be an important parameter in aggressive NHL when establishing prognosis, as well as an integral part in the pathophysiology of the disease process. While many aggressive B cell NHLs respond initially to chemotherapeutic regimens such as CHOP-bleo (adriamycin, vincristine and bleomycin) etc., relapse is common, and the patient is then often refractory to further salvage treatment regimens.^ To assess their potential to inhibit aggressive B cell NHLs and induce apoptosis (also referred to as programmed cell death (PCD)), it was proposed to utilize the following biological agents-liposomal all-trans retinoic acid (L-ATRA) which is a derivative of Vitamin A in liposomes and Vitamin D3. Preliminary evidence indicates that L-ATRA may inhibit cell growth in these cells and may induce PCD as well. Detailed studies were performed to understand the above phenomena by L-ATRA and Vitamin D3 in recently established NHL-B cell lines and primary cell cultures. The gene regulation involved in the case of L-ATRA was also delineated. ^

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

Molecular studies of human high grade B cell non -Hodgkin's lymphomas

The non-Hodgkin's B cell lymphomas are a diverse group of neoplastic diseases. The incidence rate of the malignant tumors has been rising rapidly over the past twenty years in the United States and worldwide. The lack of insight to pathogenesis of the disease poses a significant problem in the early detection and effective treatment of the human malignancies. These studies attempted to investigate the molecular basis of pathogenesis of the human high grade B cell non-Hodgkin's lymphomas with a reverse genetic approach. The specific objective was to clone gene(s) which may play roles in development and progression of human high grade B cell non-Hodgkin's lymphomas.^ The messenger RNAs from two high grade B cell lymphoma lines, CJ and RR, were used for construction of cDNA libraries. Differential screening of the derived cDNA libraries yielded a 1.4 kb cDNA clone. The gene, designated as NHL-B1.4, was shown to be highly amplified and over-expressed in the high grade B cell lymphoma lines. It was not expressed in the peripheral blood lymphoid cells from normal donors. However, it was inducible in peripheral blood T lymphocytes by a T cell mitogen, PHA, but could not be activated in normal B cells by B cell mitogen PMA. Further molecular characterization revealed that the gene may have been rearranged in the RR and some other B cell lymphoma lines. The coding capacity of the cDNA has been confirmed by a rabbit reticulocyte lysate and wheat germ protein synthesis system. A recombinant protein with a molecular weight of approximate 30 kDa was visualized in autoradiogram. Polyclonal antisera have been generated by immunization of two rabbits with the NHL-B1.4 recombinant protein produced in the E. coli JM109. The derived antibody can recognize a natural protein with molecular weight of 49 kDa in cell lysate of activated peripheral T lymphocytes of normal donors and both the cell lysate and supernatant of RR B cell lymphoma lines. The possible biologic functions of the molecule has been tested preliminarily in a B lymphocyte proliferation assay. It was found that the Q-sepharose chromatograph purified supernatant of COS cell transfection could increase tritiated thymidine uptake by B lymphocytes but not by T lymphocytes. The B cell stimulatory activity of the supernatant of COS cell tranfection could be neutralized by the polyclonal antisera, indicating that the NHL-B1.4 gene product may be a molecule with BCGF-like activity.^ The expression profiles of NHL-B1.4 in normal and neoplastic lymphoid cells were consistent with the current B lymphocyte activation model and autocrine hypothesis of high grade B cell lymphomagenesis. These results suggested that the NHL-B1.4 cDNA may be a disease-related gene of human high grade B cell lymphomas, which may codes for a postulated B cell autocrine growth factor. ^

The role of Ski/Sno oncoproteins as negative regulators of the TGF-beta/SMAD signaling pathway in B -cell non -Hodgkin's lymphoma (NHL-B)

Non-Hodgkin's lymphomas are common tumors of the human immune system, primarily of B cell lineage (NHL-B). Negative growth regulation in the B cell lineage is mediated primarily through the TGF-β/SMAD signaling pathway that regulates a variety of tumor suppressor genes. Ski was originally identified as a transforming oncoprotein, whereas SnoN is an isoform of the Sno protein that shares a large region of homology with Ski. In this study, we show that Ski/SnoN are endogenously over-expressed both in patients' lymphoma cells and NHL-B cell lines. Exogenous TGF-β1 treatment induces down-regulation of Ski and SnoN oncoprotein expression in an NHL-B cell line, implying that Ski and SnoN modulate the TGF-β signaling pathway and are involved in cell growth regulation. Furthermore, we have developed an NHL-B cell line (DB) that has a null mutation in TGF-β receptor type II. In this mutant cell line, Ski/SnoN proteins are not down-regulated in response to TGF-β1 treatment, suggesting that downregulation of Ski and SnoN proteins in NHL-B require an intact functional TGF-β signaling pathway Resting normal B cells do not express Ski until activated by antigens and exogenous cytokines, whereas a low level of SnoN is also present in peripheral blood Go B cells. In contrast, autonomously growing NHL-B cells over-express Ski and SnoN, implying that Ski and SnoN are important cell cycle regulators. To further investigate a possible link between reduction of the Ski protein level and growth inhibition, Ski antisense oligodeoxynucleotides were transfected into NHL-B cells. The Ski protein level was found to decrease to less than 40%, resulting in restoring the effect of TGF-β and leading to cell growth inhibition and G1 cell cycle arrest. Co-immunoprecipitation experiments demonstrated that Ski associates with Smad4 in the nucleus, strongly suggesting that over-expression of the nuclear protein Ski and/or SnoN negatively regulates the TGF-β pathway, possibly by modulating Smad-mediated tumor suppressor gene expression. Together, in NHL-B, the TGF-β/SMAD growth inhibitory pathway is usually intact, but over-expression of the Ski and/or SnoN, which binds to Smad4, abrogates the negative regulatory effects of TGF-β/SMAD in lymphoma cell growth and potentiates the growth potential of neoplastic B cells. ^

Dysregulated CD40-NF-kappaB signaling in the pathophysiology of aggressive non -Hodgkin's lymphoma B cells

Non-Hodgkin's Lymphomas (NHL) are a group (>30) of important human lymphoid cancers that unlike other tumors today, are showing a marked increase in incidence. The lack of insight to the pathogenesis of B-cell NHL poses a significant problem in the early detection and effective treatment of these malignancies. This study shows that large B-cell lymphoma (LBCL) cells, the most common type of B-cell NHL (account for more than 30% of cases), have developed a novel mechanism for autonomous neoplastic B cell growth. We have identified that the key transcription factor NF-κB, is constitutively activated in LBCL cell lines and primary biopsy-derived LBCL cells, suggesting that they are autonomously activated, and do not require accessory T-cell signaling for cell growth and survival. Further studies have indicated that LBCL cells ectopically express an important T-cell associated co-mitogenic factor, CD154 (CD40 ligand), that is able to internally activate the CD401NF-κB pathway, through constitutive binding to its cognate receptor, CD40, on the lymphoma cell surface. CD40 activation triggers the formation of a “Signalosome” comprising virtually the entire canonical CD40/NF-κB signaling pathway that is anchored by CD40 in plasma membrane lipid rafts. The CD40 Signalosome is vulnerable to interdiction by antibody against CD40 that disrupts the Signalosome and induces cell death in the malignant cells. In addition to constitutive NF-κB activation, we have found that the nuclear factor of activated T cells (NFAT) transcription factor is also constitutively activated in LBCL cells. We have demonstrated that the constitutively active NFATc1 and c-rel members of the NFAT and NF-κB families of transcription factors, respectively, interact with each other, bind to the CD154 promoter, and synergistically activate CD154 gene transcription. Down-regulation of NFATc1 and c-rel with small interfering RNA inhibits CD154 gene transcription and lymphoma cell growth. Our findings suggest that continuous CD40 activation not only provides dysregulated proliferative stimuli for lymphoma cell growth and extended tumor cell survival, but also allows continuous regeneration of the CD40 ligand in the lymphoma cell and thereby recharges the system through a positive feedback mechanism. Targeting the CD40/NF-κB signaling pathway could provide potential therapeutic modalities for LBCL cells in the future. ^