Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium.

Cell-cell communication is essential for tissue homeostasis, but its contribution to disease prevention remains to be understood. We demonstrate the involvement of connexin 43 (Cx43, also known as GJA1) and related gap junction in epithelial homeostasis, illustrated by polarity-mediated cell cycle entry and mitotic spindle orientation (MSO). Cx43 localization is restricted to the apicolateral membrane of phenotypically normal breast luminal epithelial cells in 3D culture and in vivo Chemically induced blockade of gap junction intercellular communication (GJIC), as well as the absence of Cx43, disrupt the apicolateral distribution of polarity determinant tight junction marker ZO-1 (also known as TJP1) and lead to random MSO and cell multilayering. Induced expression of Cx43 in cells that normally lack this protein reestablishes polarity and proper MSO in 3D culture. Cx43-directed MSO implicates PI3K-aPKC signaling, and Cx43 co-precipitates with signaling node proteins ß-catenin (CTNNB1) and ZO-2 (also known as TJP2) in the polarized epithelium. The distribution of Cx43 is altered by pro-inflammatory breast cancer risk factors such as leptin and high-fat diet, as shown in cell culture and on tissue biopsy sections. The control of polarity-mediated quiescence and MSO may contribute to the tumor-suppressive role of Cx43.

Hepatitis A virus in the Middle East and North Africa region: a new challenge.

During the past three decades, a gradual shift in the age of infection with hepatitis A virus (HAV) from early childhood to adulthood has been observed. There is a general lack of updated data on HAV burden of disease, incidence and age-specific seroprevalence in countries of the Middle East and North Africa (MENA) region. The aim of this article is to review the published data on anti-HAV seroprevalence, an important tool to monitor infections rates, in countries of the MENA region and associated risk factors including water and socioeconomic data when available. Data on anti-HAV seroprevalence were found for 12 of 25 MENA countries. We show that MENA countries, similar to other areas in the world, have a clear shift in HAV incidence with a decline among young age groups and an increase among adults and older individuals. This would likely be associated with increased morbidity and increased risks of outbreaks among younger age groups. Consequently, the continuous surveillance of hepatitis A cases and the inclusion of hepatitis A vaccine in the expanded immunization programmes are needed in countries of the MENA.

Connexins and the gap in context.

Gap junctions (GJ) can no longer be thought of as simple channel forming structures that mediate intercellular communication. Hemi-channel and channel-independent functions of connexins (Cxs) have been described and numerous Cx interacting partners have been uncovered ranging from enzymes to structural and scaffolding molecules to transcription factors. With the growing number of Cx partners and functions, including well-documented roles for Cxs as conditional tumor suppressors, it has become essential to understand how Cxs are regulated in a context-dependent manner to mediate distinct functions. In this review we will shed light on the tissue and context-dependent regulation and function of Cxs and on the importance of Cx-interactions in modulating tissue-specific function. We will emphasize how the context-dependent functions of Cxs can help in understanding the impact of Cx mis-expression on cancer development and, ultimately, explore whether Cxs can be used as potential therapeutic targets in cancer treatment. In the end, we will address the need for developing relevant assays for studying Cx and GJ functions and will highlight how advances in bioengineering tools and the design of 3D biological platforms can help studying gap junction function in real time in a non-intrusive manner.

Anti-inflammatory bioactivities in plant extracts.

The medical ethnobotanical knowledge propagated over generations in the coastal regions of the Eastern Mediterranean, including Lebanon, is one that has built on several ancient cultures and civilizations of these regions. Recent interest in medical ethnobotany and the use of medicinal herbs in treating or preventing ailments has rejuvenated interest in folk medicine practices, especially those transcendent across generations. According to Eastern Mediterranean folk medicine practices, herbal remedies that treat many inflammation-related ailments were typically based on plant bioactive water extracts or decoctions. Studies have shown that active anti-inflammatory ingredients in water extracts include many natural chemicals such as phenols, alkaloids, glycosides, and carbohydrates. The intent of this manuscript is twofold: first, to review the literature that describes anti-inflammatory bioactivities in plant extracts of different plant genera; and second, to evaluate indigenous folk remedies used by folk doctors to treat inflammatory ailments in this region of the world. For this aim, the reported literature of five plant genera assumed to possess anti-inflammatory bioactivities and typically prescribed by folk doctors to treat inflammation-related ailments is reviewed.

The effect of endotoxin on functional parameters of mammary CID-9 cells.

The effect of endotoxin on mammary CID-9 cells, which differentiate in culture and express beta-casein, was investigated. Cells in culture supplemented with lactogenic hormones and dripped with EMS-Matrix (EMS-drip), were treated daily with endotoxin (0.5-500 microg/ml). Endotoxin at concentrations of less or equal to 10 microg/ml did not affect cell growth and viability up to 5 days post endotoxin treatment. Endotoxin (0.01-10 microg/ml) was added to the culture medium, upon confluence, and functional parameters were examined within 48 h post endotoxin treatment. Nuclear factor-kappaB (NF-kappaB) (p52) increased in nuclear extracts from endotoxin-stimulated cells within 1 h of treatment, while beta-casein mRNA and protein expression decreased in a concentration-dependent manner at 24 and 48 h post treatment. Zymography showed that the 72 and 92 kDa gelatinase activity increased in cells at 24 and 48 h post endotoxin treatment at 10 and 50 microg/ml. At the latter concentration, the active form of 72 kDa gelatinase was induced at 48 h. Interleukin-6 and tumor necrosis factor-alpha levels increased at 1-3 h post endotoxin treatment and peaked at 6 h in cells on plastic and EHS-drip. Nerve growth factor (NGF) levels increased in control and endotoxin-treated cells in a time-dependent manner, and endotoxin increased NGF levels in culture at 6 and 9 h post endotoxin treatment. This study shows that endotoxin activated NF-kappaB, suppressed beta-casein expression and upregulated gelatinases, cytokines and NGF. This model could be used to investigate the role of mammary cells in initiating and propagating inflammation and to test candidate molecules for potential anti-inflammatory properties.

Functional consequence of exposure to dieldrin on mammary development and function.

The effect of dieldrin (Dln) on the development of the mammary gland and on functional parameters of CID-9 mammary cells in culture was investigated. One-month-old Sprague-Dawley female rats were bred and received intraperitoneal injection with 2.5 or 15 microM Dln during the last trimester of their gestation. Mammary glands of 15-microM Dln-treated rats showed immature alveolar structures by day 18 of gestation and abundant adipose tissue. Dln-treated rats had a lower number of pups, and the weight of pups between days 14 and 31 of age compared with non-treated rats was significantly lower. Long-term exposure of CID-9 mammary cells, cultured under non-differentiation conditions, on plastic, or under differentiation permissive conditions, dripped with EHS-matrix, to 5 or 25 microM Dln was detrimental to cell growth. The short-term effect of Dln exposure (up to 9 h) on CID-9 cells, under the same culture conditions, did not affect their beta-casein mRNA levels, but induced apoptosis, down regulated gap junction intracellular communication and induced IL-6 and TNF-alpha expression.

Partial purification and characterization of proteins with growth promoting activities from ovine mammary gland secretions.

Developmental regulation of growth promoting activities in mammary secretions of pregnant Awassi ewes was defined, and growth factors contained in these secretions were partially purified and characterised. Mammary secretions from pregnant ewes enhanced fibroblast cell (AKR-2B) and mammary cell (CID-9 cell strain) proliferation to levels comparable to that induced by 10% Foetal calf serum. Major milk proteins in mammary secretions collected from pregnant ewes one month prior to lambing up to one week after lambing, were resolved by SDS-PAGE, while gelatinases were resolved by zymography. Gelatinase activity was noted prior to P134 and decreased thereafter to reach a minimum during lactation. This decrease was concomitant with the onset of casein production. It is during this critical developmental period that highest growth promoting activity in mammary secretions was detected. Secretions with highest growth promoting activity were fractionated by ion exchange and gel filtration chromatography. Two heat-resistant, trypsin/chymotrypsin sensitive, growth-promoting activities were characterised. The first, designated ovine mammary derived growth factor-1 (oMDGF-1), had around a 30 kDa molecular weight and eluted at 0.65 M NaCl gradient on cation ion exchange chromatography. The second, oMDGF-2, eluted under gel filtration conditions at a molecular weight of 50 kDa and 150 kDa. oMDGF-1 induced changes in Connexin 43, but not in beta-casein mRNA expression by CID-9 mammary cells. In conclusion, growth factor activities in ewe mammary secretions peak during gestation at a period that overlaps maximal gelatinase expression and precedes milk protein synthesis. The factors modulate mammary cell function and may play a role in mammary gland development.

Comparative performance of PCR-based assay versus microscopy and culture for the direct detection of Mycobacterium tuberculosis in clinical respiratory specimens in Lebanon.

SETTING: American University of Beirut Medical Center, Lebanon. OBJECTIVE: To assess the performance of a polymerase chain reaction (PCR) using primers that flank 542 bp within IS6110 in Mycobacterium tuberculosis (TB) vs. microscopy and BACTEC culture, in the diagnosis of tuberculosis. DESIGN: A total of 82 clinical respiratory pulmonary specimens and 73 samples from BACTEC vials were tested by the three methods. RESULTS: Of 24 smear-positive culture-positive (SP-CP) and 11 smear-negative culture-positive (SN-CP) TB specimens, PCR detected 83% and 64%, respectively. Among 17 specimens yielding mycobacteria other than tuberculosis (MOTT), the PCR was positive in 33% SP-CP and 14% SN-CP specimens. Among the 73 BACTEC vials, PCR was positive in 36 of 38 (95%) yielding culture-positive TB, and in one of 20 (5%) yielding culture positive MOTT. None of the 30 smear-negative culture-negative (SN-CN) clinical specimens and 15 of the CN vials were positive by PCR. The overall sensitivity of PCR was 77% and 95% for TB detection in respiratory specimens and BACTEC vials, respectively, and the specificity was 94% in both. CONCLUSIONS: Because a substantial number of TB cases are missed, especially in SN-CP specimens, a PCR-based assay utilizing these primers cannot be used reliably, alone, in clinical laboratory diagnosis of mycobacterial respiratory infections.

Functional interplay between gelatinases and hyperalgesia in endotoxin-induced localized inflammatory pain.

The role of ECM-degrading proteinases in normal developmental processes and in pathological conditions is extensively studied. However, few reports describe the role ECM-degrading proteinases play in modulating hyperalgesia. The goal of this study is to describe the regulation of gelatinases during endotoxin mediated local inflammation, induced by intra plantar endotoxin (ET; 1.25 microg/50 microl) injection in Balb/c mice, and to correlate that with hyperalgesia. ET injections induced hyperalgesia, as determined by hot plate and paw pressure tests, which peaked by 24 h and recovered by 48 h post-injection. Contralateral paw of ET injected mice and saline injected paws in control mice elicited no hyperalgesia. Zymography showed that ET and saline injected paws elicited increased gelatinase activity by 9 h after injection. However, only the former maintained high levels of expression of a 90 kD gelatinase up to at least 96 h post ET injection, while in the latter gelatinase expression was down regulated by 24 h. Interestingly, the 90-kD gelatinase was upregulated in the contralateral paw of the ET-injected mice beyond 48 h post injection. Saline injection in that paw, during a time when gelatinases are upregulated, induced hyperalgesia. Intraperitoneal injection of either ZnCl(2) (100 microM), thymulin (5 microg/100 microl), or morphine (2 mg/kg/100 microl) reversed the ET-induced hyperalgesia and suppressed gelatinase activity. Furthermore, intraperitoneal injection of MPI, an ECM-degrading proteinase inhibitor, reversed ET induced hyperalgesia. Taken together, the above suggests that a functional interplay exists between gelatinase upregulation triggered by ET injections and hyperalgesia. The exact mechanism underlying such correlation remains to be determined.

Emergence of Salmonella enteritidis outbreaks in broiler chickens in the Lebanon: epidemiological markers and competitive exclusion control.

This study investigates the first emergence of Salmonella Enteritidis outbreaks among chickens in the Lebanon and identifies the epidemiological markers of selected recovered Enteritidis strains. In addition, the authors evaluate a competitive exclusion approach to control infection in broiler chickens by Enteritidis organisms which possess the prevalent identified markers. The basic procedure in this investigation involved recording signs and lesions in eleven broiler chicken flocks on eleven farms, and culturing livers, spleens, and caeca of ten randomly selected birds per flock for Salmonella isolation and serotyping. Furthermore, culturing for Salmonella and serotyping was attempted from the livers, spleens, caeca and oviduct swabs of ten hens in four broiler breeder flocks which provided hatching eggs for the broilers under study. The identification of epidemiological markers in recovered S. Enteritidis included the determination of drug-resistance patterns and plasmid profiling. The competitive exclusion was evaluated by spraying the microflora on day-old broilers in the hatchery, followed by a controlled oral challenge at three days of age, with 2.85 x 10(5) colony-forming units of S. Enteritidis organisms per bird. Exclusion was evaluated by culturing for S. Enteritidis in anal swabs, spleens, livers, and caeca of individual challenged birds treated with the microflora and in untreated challenged birds. A total of 112 invasive S. Enteritidis strains were recovered on eleven farms from individual organs of broiler chickens with typical signs and lesions of salmonellosis. The prevalent resistance to drugs in such strains was to furaltadone and gentamycin, a marker identified in 93 strains (83%), recovered from nine out of eleven farms. The same resistance pattern was present in S. Enteritidis strains recovered from breeders on one out of four farms. The prevalent plasmid profile in nine S. Enteritidis organisms selected randomly from a pool of 93 strains (one per each of the nine broiler farms) was 14.1 kilobases (kb) and approximately 50.0 kb, a typical pattern to that identified in S. Enteritidis organisms recovered from oviducts of breeders on one out of four breeder farms. The exclusion significantly reduced cumulative mortality in birds of up to 45 days of age by 3.93%, in comparison to that observed in untreated challenged birds (P < 0.05). At 45 days of age, exclusion resulted in a 15.6% reduction in the percentage infection rate by S. Enteritidis in spleens or livers and a 34.4% reduction in the percentage infection rate of the caeca (P < 0.05).

Discrepancies between mecA PCR and conventional tests used for detection of methicillin resistant Staphylococcus aureus.

Conventional and molecular techniques are being used in the detection of methicillin resistance in Staphylococcus aureus but they do not always show concordant results. In this study, a mecA PCR-based amplification was compared with the 1 microg oxacillin disk diffusion test and the Epsilometer test (E-test) for detection of MICs. Among 31 isolates initially characterized as MRSA by the disk diffusion test, mecA was detected in only 13 (42%) isolates. The E-test showed a wide range of oxacillin MICs (0.5 - > 256 microg/ml) among these 31 MRSA isolates: seven isolates had an MIC of > 256 microg/ml, one had 64 microg/ml, two had 4 microg/ml, two had 3 microg/ml, one had 2.5 microg/ml, nine had 2 microg/ml, three had 1.5 microg/ml, five had 1 microg/ml and one had 0.5 microg/ml. Comparing the mecA PCR results with the E-test oxacillin MIC findings revealed that mecA was detected in seven of eight isolates (87.5%) with an MIC of > or = 64 microg/ml, in three of 14 isolates (21.4%) with an MIC of 2-4 microg/ml and in three of nine isolates (33.3%) with an MIC of < 2 microg/ml. Beta-lactamase production was positive in 28/31 isolates (90.3%). Because of this variation between tests and since several resistance mechanisms are known to mediate methicillin resistance in S. aureus, the reliable detection of MRSA cannot be solely based on detection of mecA gene in S. aureus. At this stage and until new guidelines are introduced by an official body, such as NCCLS, a combination of conventional methods alone or together with a molecular method should be used every time S. aureus is tested for detection of methicillin resistance.

Relationship of dietary intake to DDE residues in breast milk of nursing mothers in Beirut.

Milk samples were collected from 32 nursing mothers living in the Beirut area, Lebanon. Dietary intakes of participating mothers were obtained from data of their diet histories, 24 h dietary recalls and food frequency questionnaires. Milk samples were screened for the presence of organochlorine pesticide residues and DDE levels were estimated using gas chromatographic techniques. The relationship between consumption of various food groups and DDE content of milk was investigated. A positive correlation was found between the consumption of either/or high fat meat, tuna fish and DDE levels in milk. Consumption of poultry products showed a weak correlation with DDE content of milk, whereas consumption of vegetable oils showed a negative correlation.

Effect of substratum on growth, cell morphology and lactoferrin synthesis and secretion in bovine mammary cell culture.

The role of extracellular matrix in morphology, growth and lactoferrin synthesis and secretion in bovine mammary cells from a developing gland is poorly defined. In this study, bovine mammary cells from a hormone-primed developing gland were isolated and cultured on plastic, collagen, embedded within collagen, or on EHS-matrix, with the hormones prolactin, insulin, and cortisol in the presence or absence of fetal calf serum. Mammary cells on plastic or collagen spread and formed confluent cells sheets, while those embedded within collagen or on EHS-matrix maintained their acinar-like structure. Histological and ultrastructural analysis of cells showed that cells on plastic and collagen grew in multilayers, while those embedded within collagen or on EHS-matrix lacked any lumen structure. The ultrastructure of cells on different substrata more resembled an undifferentiated phenotype. Mammary cells secreted lactoferrin in increasing concentrations throughout the culture period. The total amount secreted in culture was regulated by extracellular matrix and fetal calf serum. Cells embedded within collagen in serum-free cultures secreted the lowest amounts of lactoferrin (up to 619 ng/ml; day 14), while those on collagen and supplemented with fetal calf serum secreted up to 4920 ng/ml at day 14. Fetal calf serum induced higher lactoferrin secretion within each substratum on which the cells were cultured. No intracellular accumulation of lactoferrin was noted in cells on plastic or collagen or those embedded within collagen, whereas those on EHS-matrix accumulated more than 500 ng/ml of lactoferrin intracellularly/intracinarly. Furthermore, when cultured on a similar substratum, cells from a developing gland secreted higher lactoferrin than cells from a lactating gland.

Evaluation of an enrofloxacin-treatment program against Mycoplasma gallisepticum infection in broilers.

Three chicken broiler breeder flocks, 7 months of age, were confirmed to have Mycoplasma gallisepticum (MG) infection, based on culture of tracheal swabs. A total of fifty-five 7-day-old embryos from the three MG-positive flocks had an average 27.4% prevalence of MG-infection in their vitelline membrane. Sixty randomly selected MG isolates (30 from individual tracheas of breeders and another 30 from individual vitelline membrane of embryos) were highly sensitive in vitro to enrofloxacin (100%). Three broiler flocks (averaging 15,000 birds per flock) from the same three MG-infected chicken boiler breeders were divided into halves. The first halves were subjected to an enrofloxacin-treatment program and the other halves were controls. Sera collected at different ages of the broiler flocks were tested by the enzyme-linked immunosorbent assay for antibodies to MG. The absence of MG titers at 45 days of age in birds subjected to the enrofloxacin-preventive program was compared to an average prevalence of 15.9% in the controls (p < 0.05). The lack of MG titers in 45-day-old birds subjected to the enrofloxacin-treatment program was associated with lower better feed-conversion ratios (p < 0.05).

Prevalence, antimicrobial susceptibility and molecular characterization of Campylobacter isolates recovered from humans and poultry in Lebanon.

Recovery of Campylobacter was attempted from 281 consecutive non selected out-patients diarrheic stools, 150 individual ceca collected from meat chicken breeder farms and 31 slaughtered marketed chicken obtained from shops in Lebanon. Campylobacter isolates were recovered from 2 (0.7%) human stool specimens, 34 (22.7%) chicken ceca and 3 (9.7%) raw chicken carcasses. Speciation of these isolates revealed 2 C. jejuni from humans diarrheic stools, 16 C. coli, 10 C. jejuni, 3 C. fetus, 2 C. fennelliae (Helicobacter fennelliae, new taxon), 2 C. upsaliensis, 1 C. cryaerophila (Archobacter cryaerophilus, new taxon) from chicken ceca and 2 C. coli and 1 C. fennelliae (H. fennelliae) from raw chicken carcasses. Antimicrobial susceptibility testing of the different isolates against 9 antimicrobial agents was performed using the E-test. Overall, most isolates showed high to moderate susceptibility to gentamicin (97%), amoxicillin/clavulanate (95%), clindamycin (77%), chloramphenicol (77%), and ampicillin (69%). Lower susceptibility were observed against tetracycline (49%), erythromycin (47%), ciprofloxacin (39%), and norfloxacin (36%). This overall susceptibility profile generally applied to C. coli and C. jejuni, as well, although C. coli mostly showed higher susceptibility than C. jejuni. beta-lactamase production was detected in 59% of all the isolates, being higher in C. coli (72%) than C. jejuni (33%). Whole cell protein profile analysis of 18 C. coli and 12 C. jejuni by SDS-PAGE revealed 6 different patterns. In both species, major variations existed in the region between mol wt 45-60 and all protein profiles were dominated by the presence of 5 major bands of mol wt: 61 (doublet), 45, 31 and an approximate 24. Differences in banding patterns within and between both species indicated diversity and heterogeneity of strains. This study shows that despite high prevalence and diversity of strains in chicken, Campylobacter in Lebanon is rare in human diarrheic stools compared to Salmonella (3.2%) and Shigella (1.4%).

The spectrum of beta-thalassaemia mutations in the Lebanon.

We screened 110 DNA samples from carriers of beta-thalassaemia, using the ARMS-PCR technique with primers for common Mediterranean mutations. Unidentified samples were subjected to a heteroduplex analysis with Universal Heteroduplex Generators covering the beta-globin gene, followed by DNA sequencing. In total, 16 different mutations were detected, the most frequent being IVSI-110 (40%), followed by other common Mediterranean mutations (IVSI-1, IVSII-1, IVSI-6). Other mutations detected were of Lebanese, Turkish, Iranian, Kurdish, Bulgarian and Asian Indian origin. The most heterogeneous religious group seems to be the Sunni Muslims, with 13 mutations, while only 2 mutations were detected among the Christian Maronites. Results from this study are compared with those from other Mediterranean and neighbouring countries.

Developmental regulation and partial characterization of growth factors in the bovine mammary gland.

Bovine mammary gland secretions from several developmental stages were shown to stimulate [3H]thymidine incorporation into AKR-2B mouse embryo fibroblast cells. In virgin heifers, mammary secretions at 1% concentration stimulated thymidine incorporation into AKR-2B cells more than threefold compared with 10% (v/v) fetal calf serum. The growth-promoting activity peaked at an early stage of the last trimester (7-8 months) of gestation and declined after this until the colostrum forming stage. At this point, the activity was one- to twofold that induced by 10% (v/v) fetal calf serum. At parturition the activity dropped abruptly to virtually undetectable values in milk. The developmental change in activity could be mimicked by hormonal priming of the mammary gland. The partial characterization of the growth promoting activities in secretions at the seventh month of gestation revealed at least two major growth-promoting activities: bovine mammary derived growth factor 1 (bMDGF-1), an epidermal growth factor-like growth factor with a molecular weight of 30,000, which is trypsin sensitive and heat stable, and bMDGF-2, which eluted under gel filtration conditions at a molecular weight of 50,000 and 150,000. bMDGF-1 is predominant in pregnant, precolostric, and colostric secretions, and is not detected in milk. bMDGF-2 is the major growth factor in milk. These results show developmental regulation and modulation of growth-promoting factors during the different stages of mammary gland development and suggest that growth factors are involved in regulating growth during gestation.