Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Winged leaf-cutting ants on nuptial flights used as transport by Attacobius spiders for dispersal

Abstract.  1. Sexuals of a leaf-cutting ant, Atta bisphaerica Forel, left their nest for nuptial flights in October to December.
2. When leaving a nest, 53 of the 479 winged sexuals (or alates) observed (11.1%) carried up to three inquiline spiders of Attacobius luederwaldti .
3. Spiders exclusively selected winged sexuals, not workers, and preferred females, indicating their expectation of the stronger flight ability of females. Neither these sexuals nor workers that appeared out of the nest on flight days attempted to remove or attack spiders on the body of alates.
4. New qucens landing from their nuptial flight did not carry spiders, indicating that the spiders had left the ants in the sky to be dispersed by wind.
5. No spiders were found in more than 100 incipient nests, which were estimated to be 2–3 months old. This suggests that the spiders jumped off the alate during mid-flight and dispersed on the wind to inhabit larger nests.     

Protective role of potentially lethal damage repair in the neoplastic transformation of Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

To determine the role of repair of potentially lethal damage (PLD) in the initiation process of neoplastic transformation, Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were temporarily exposed to conditioned medium obtained from density-inhibited Chinese hamster cell cultures, as a post-treatment for the induction of PLD repair. With or without this exposure, cell survival and transformation frequencies were simultaneously determined by colony-formation and focus-formation assays, respectively. Temporary exposure to conditioned medium resulted in a 20-30% increase in cell survival compared with no exposure. Post-treatment with conditioned medium resulted in a 60-65% reduction in transformation frequencies. At the molecular level, the repair of MNNG-induced single-strand breaks of DNA occurred much more rapidly in conditioned medium. These data suggest that PLD repair reduces the in vitro neoplastic transformation through excision repair operative during the cessation of DNA replication. Thus, PLD repair appears to be preventive against neoplastic fixation in initiation of neoplastic development.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Destruction of tumor cells by monokines released from activated human blood monocytes: evidence for parallel and additive effects of IL-1 and TNF

Summary The incubation of human peripheral blood monocytes with endotoxins activates the cells to lyse tumorigenic targets directly and also induces the production and release into the culture medium of factors that produce lysis of mouse-transformed fibroblasts L-929 (tumor necrosis factor (TNF)-sensitive) and human A-375 melanoma cells (interleukin-1 (IL-1)- and TNF-sensitive). Immunoblotting analysis revealed that the culture medium of endotoxin-activated but not of control monocytes contained both IL-1 and TNF with a molecular weight of 17,000 daltons each. TNF activity was determined by lysis of L-929 cells, and IL-1 activity was measured by the proliferation of D-10 cells. The production of IL-1 and TNF was concentration-dependent, and the amounts of these monokines were paralleled. The antitumor activity of the culture supernates from endotoxin-treated monocytes was significantly decreased by incubation with heterologous antisera to IL-1, TNF, or both. Recombinant human IL-1 and TNF were used in parallel experiments and as positive controls. Each monokine used produced cytotoxic effects in susceptible targets. The combination of IL-1 and TNF, which more likely resembles culture supernates of activated macrophages, produced an additive antitumor cytotoxicity effect.     

Tumor necrosis factor and IL-1 associated with plasma membranes of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both

The purpose of our study was to determine some of the mechanisms involved in macrophage-mediated lysis of tumorigenic cells. A375 human melanoma cells (A375-R) resistant to lysis mediated by TNF and IL-1 were selected from the TNF- and IL-1-sensitive A375 parental melanoma cells subsequent to continuous (2 mo) exposure to rTNF. Peripheral blood monocytes isolated by centrifugal elutriation from healthy donors were incubated with rIFN-gamma and muramyl dipeptide, with a lipoprotein derived from Escherichia coli (CG-31362) or with LPS for 24 h. These activated monocytes lysed both the A375 (monokine-sensitive) and A375-R (monokine-resistant) melanoma cells. Activated tumoricidal macrophages fixed in 2% paraformaldehyde lysed only the TNF- and IL-1-sensitive A375 cells. These fixed monocytes contained both IL-1 and TNF activities as determined by D10 cell proliferation and L929 cytolysis assays, respectively. Nearly identical results were obtained with preparations of plasma membranes from activated human monocytes. Anti-IL-1 and/or anti-TNF sera neutralized the cytolysis of tumor cells mediated by free monokines, by fixed monocytes, or by plasma membrane preparations. In contrast, anti-TNF and/or anti-IL-1 sera did not inhibit tumor cell lysis by viable activated monocytes. We conclude that IL-1 and TNF molecules associated with the plasma membranes of activated monocytes mediate lysis of susceptible target cells. However, because activated monocytes lysed IL-1-and TNF-resistant target cells, molecules other than these monokines must also be involved in the antitumor activity of monocytes.     

Augmentation of therapeutic effect of adoptive immunotherapy through a synergy between transferred killer cells and host's fresh lymphocytes]

Among several approaches to augment the therapeutic effect of adoptive immunotherapy, we focused the antitumor synergy between transferred killer cells and host's fresh lymphocytes. Immunotherapy models using murine tumors or clinical experiments revealed that preadministration of immunostimulator such as OK-432, followed by chemotherapeutic agents such as cyclophosphamide, can induce host's non-cytotoxic fresh lymphocytes that act synergistically with cultured killer cells against autologous tumor cells. Immuno-chemo-lymphocytotherapy (a sequential treatment with OK-432, chemotherapy and adoptive immunotherapy) is useful to treat the patients with advanced cancer even if the number of transferred lymphocytes is limited.     

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)