The human imperative of stabilizing global climate change at 1.5°C.

Increased concentrations of atmospheric greenhouse gases have led to a global mean surface temperature 1.0°C higher than during the pre-industrial period. We expand on the recent IPCC Special Report on global warming of 1.5°C and review the additional risks associated with higher levels of warming, each having major implications for multiple geographies, climates, and ecosystems. Limiting warming to 1.5°C rather than 2.0°C would be required to maintain substantial proportions of ecosystems and would have clear benefits for human health and economies. These conclusions are relevant for people everywhere, particularly in low- and middle-income countries, where the escalation of climate-related risks may prevent the achievement of the United Nations Sustainable Development Goals.

The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum. Immunological localization, cloning and sequencing of its gene porA.

The 5-kDa protein PorA of the Gram-positive bacterium Corynebacterium glutamicum is the subunit of the cell wall channel. Antibodies raised against PorA specifically detected the protein on the cell surface. PorA was sequenced using Edman degradation and a gas phase sequencer. The primary sequence was used to create degenerate oligonucleotide primers. The gene of the channel-forming protein and its flanking regions were obtained by PCR followed by inverse PCR. The gene porA comprises 138 bp and encodes a 45-amino-acid-long acidic polypeptide with an excess of four negatively charged amino acids in agreement with the high cation selectivity of the PorA cell wall channel. PorA does not contain an N-terminal extension. A ribosomal-binding site was recognized 6 bp before the start codon ATG of porA. It codes for the smallest subunit of a membrane channel known so far and for the first cell wall channel protein of a corynebacterium. Southern blots demonstrated that only the chromosomes of corynebacteria contain homologous sequences to porA; no hybridization could be detected with DNA from other mycolata.

Analysis of IgG isotype responses against Onchocerca volvulus antigens in children in an onchocerciasis-endemic area in northern Nigeria.

This study evaluated the usefulness of parasite-specific IgG and IgG isotype responses for diagnosing early or pre-patent onchocerciasis in children using sandwich ELISA. The children (n = 199) were aged between 5 and 12 years and living in a meso-endemic area of northern Nigeria. Only five had detectable skin microfilariae. The mean optical density (OD) values of children from the study area were significantly higher than those of normal controls' sera (n = 10, p < 0.01), except for IgG2. There were 145 (73%) children positive for total IgG, 161 (81%) for IgG1, 68 (34%) for IgG3 and 187 (94%) for IgG4 antibodies. The mean OD values of all antibodies tended to increase with age and peaked in the 9-10-year age group, except in the case of IgG3 which peaked at 11-12 years. IgG4 proved most sensitive compared with IgG, IgG1 and IgG3 in all age groups, and was significantly different (p < 0.05). Analysis by sex showed no significant difference between the boys and girls. We conclude that IgG4 serology is a useful diagnostic tool in the assay of early or pre-patent onchocerciasis in children.

Analysis of Plasmodium falciparum infections in a village community in Northern Nigeria: determination of msp2 genotypes and parasite-specific IgG responses.

The genetic diversity of P. falciparum and multiplicity of infection has been studied in a village in Northern Nigeria at the end of the rainy season, when transmission is high. We analysed blood samples from 104 individuals aged 5-70 years by polymerase chain reaction (PCR) amplifying the gene for the merozoite surface protein MSP2 followed by genotyping based on restriction fragment length polymorphism (RFLP). 94.2% of all samples were parasite positive by PCR and over 80% of those had multiple infections. The age distribution of the average number of parasite clones present in P. falciparum infections showed an initial increase, then reached a peak multiplicity in children 8-10 years of age, and afterwards decreased significantly with age. Mean multiplicity in those 8-10-year-old children was 5.4 clones per carrier. Peak multiplicity and parasite diversity in Nigerian individuals is compared to findings from other study sites in Africa and PNG. The prevalence of IgG antibodies against the circumsporozoite protein (CSP), an indicator for malaria exposure, was over 85% in all age groups showing a high exposure of villagers to P. falciparum. OD values in ELISA were positively correlated with age. There was no correlation between the level of IgG against CSP and the multiplicity of P. falciparum infections determined by PCR of msp2. These results imply that in highly endemic areas multiplicity of infection is not directly correlated with exposure to P. falciparum.

Expression of the ggpS gene, involved in osmolyte synthesis in the marine cyanobacterium Synechococcus sp. Strain PCC 7002, revealed regulatory differences between this strain and the freshwater strain Synechocystis sp. Strain PCC 6803.

Synthesis of the osmolyte glucosylglycerol (GG) in the marine cyanobacterium Synechococcus sp. strain PCC 7002 was characterized. The ggpS gene, which encodes the key enzyme (GG-phosphate synthase [GgpS]) in GG biosynthesis, was cloned by using PCR. A 2,030-bp DNA sequence which contained one open reading frame (ORF) was obtained. The protein deduced from this ORF exhibited 85% similarity to the GgpS of the freshwater cyanobacterium Synechocystis sp. strain PCC 6803. The function of the protein was confirmed by generating a ggpS null mutant, which was not able to synthesize GG and thus exhibited a salt-sensitive phenotype. Expression of the ggpS gene was analyzed in salt-shocked cells by performing Northern blot and immunoblot experiments. While almost no expression was detected in cells grown in low-salt medium, immediately after a salt shock the amounts of ggpS mRNA and GgpS protein increased up to 100-fold. The finding that salt-induced expression occurred was confirmed by measuring enzyme activities, which were negligible in control cells but clearly higher in salt-treated Synechococcus sp. cells. The salt-induced increase in GgpS activity could be inhibited by adding chloramphenicol, while in protein extracts of the freshwater cyanobacterium Synechocystis sp. strain PCC 6803 a constitutive, high level of enzyme activity that was not affected by chloramphenicol was found. A comparison of GG accumulation in the two cyanobacteria revealed that in the marine strain osmolyte synthesis seemed to be regulated mainly by transcriptional control, whereas in the freshwater strain control seemed to be predominantly posttranslational.

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice.

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.

A novel PrfA-regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice.

Several large, cell wall-associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i-inlFE. The two proteins encoded by this locus, i-InlE and i-InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i-inlFE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inlC encodes the only small internalin. The mature i-InlE protein (198 amino acids) is secreted in large amounts into the brain-heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA-dependent gene transcription, i-inlE mRNA and i-InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i-inlF and i-inlE genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences ('PrfA-boxes') were identified in the promoter regions of i-inlF and i-inlE. Non-polar i-inlE and i-inlF deletion mutants and an i-inlFE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 109 bacteria per mouse, whereas the LD50 for the parental strain was determined as 4 x 107 bacteria per mouse. These data suggest an important role for i-InlE and i-InlF in L. ivanovii virulence.

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells.

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.

Sequence comparison of the chromosomal regions encompassing the internalin C genes (inlC) of Listeria monocytogenes and L. ivanovii.

We have recently cloned and characterized the inlC gene of Listeria monocytogenes which belongs to the listerial internalin multigene family and codes for a 30-kDa secreted protein containing five consecutive leucine-rich repeats. Here, we show that in L. monocytogenes inlC is located between the rplS gene (encoding the 50S ribosomal protein L19), and the infC gene (encoding the translation initiation factor 3). By direct and inverse polymerase chain reactions (PCR), we cloned a 5.4-kb region containing a homologous gene (termed i-inlC) from L. ivanovii, the other pathogenic member of the genus Listeria. In this microorganism, the i-inlC gene is preceded by another internalin gene, i-inlD, which seems to be specific for L. ivanovii, as this gene could not be detected in L. monocytogenes by Southern hybridization with an i-inlD gene probe. The i-inlD gene also encodes a small secretory internalin (i-InlD), which shares extended homology with (i-)InlC. Upstream of i-inlD are genes for 23S rRNA and 5S rRNA, and two tRNA genes [Asn-tDNA (GTT) and Thr-tDNA(GTT)]. The 3' terminus of the Thr-tRNA gene appears to be the site of an insertion of a genetic element including i-inlC and i-inlD. A putative transcriptional regulator gene, the product of which contains the TetR family signature, is located downstream of i-inlC. This chromosomal position of the two inlC genes on their respective chromosomes may be due to horizontal transfer of this gene. Transcription of i-inlC and i-inlD is strictly dependent on the transcriptional activator PrfA, which regulates transcription of most of the known virulence genes (including inlC) of L. monocytogenes and of L. ivanovii.

A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins.

A mutant of Listeria monocytogenes EGD was constructed that carries an extended deletion removing the entire PrfA-regulated gene cluster from plcA to plcB and a second deletion inactivating the inlA gene. Upon supplementation of this mutant with multiple gene copies of prfA, a protein of 30 kDa was detected in the supernatant of the mutant strain. The gene encoding this protein was obtained by direct and inverse polymerase chain reaction using oligonucleotide primers that were deduced from partial amino acid sequences of the purified 30 kDa protein. The amino acid sequence of the gene product revealed a protein of 297 amino acids that carried eight repeat units with high homology to those of the two known internalin proteins A and B. This secretory protein, termed internalin C, is much smaller than InlA or InlB and its complete sequence is related to the two known internalins. The gene InlC is transcribed into a monocistronic mRNA from a single promoter which shows a typical consensus sequence for PrfA-binding at the position -40. In contrast to the transcription of the InlAB operon, which is downregulated after shift of an L. monocytogenes EGD culture from brain-heart infusion into minimum essential medium (MEM), transcription of inlC is induced in MEM like most of the other known PrfA-regulated virulence genes. In addition, InlC is strongly transcribed in the cytoplasm of phagocytic J774 cells whereas inlA is poorly transcribed under these conditions, suggesting that internalin C may play a role in a late stage of L. monocytogenes infection rather than in the uptake of L. monocytogenes by non-professional phagocytic cells. An InlC deletion mutant shows reduced virulence when tested in an intravenous mouse model, but intracellular replication of the mutant in Caco-2 and J774 cells appears to be comparable with that of the wild-type strain.

Plasmodium falciparum: malaria morbidity is associated with specific merozoite surface antigen 2 genotypes.

Plasmodium falciparum merozoite surface antigen 2 (MSA2) is considered a vaccine candidate in a subunit vaccine against blood stage malaria. In order to test if a specific genotype of the highly polymorphic MSA2 is associated with disease, we conducted a case-control study in a malaria endemic area of Papua New Guinea involving 227 individuals, mostly children under the age of 10 years. All cases and controls were genotyped by polymerase chain reaction for their respective MSA2 genotypes. We report that at the time of the study parasites carrying the FC27-like genotype were twice as likely to be found in symptomatic malaria cases than in asymptomatic controls. Mixed genotype infections were significantly less frequent in symptomatic malaria infections. One individual allele (WOS10) of the FC27 family was found only in cases. This may be a form of P. falciparum infrequently encountered and may cause morbidity due to lack of immunity to it. This study provides evidence that MSA2 is involved in the morbidity of malaria and supports the inclusion of MSA2 in a subunit vaccine.

Analysis of antibody responses directed against two Onchocerca volvulus antigens defined by monoclonal antibodies.

Monoclonal antibodies, reactive with antigens solubilised from the body wall of intact female Onchocerca volvulus using 2% 2-beta-mercaptoethanol, have been characterised. Two IgG1 antibodies, Cam1 and Cam28, recognised antigens of apparent molecular weights of 18,000 and 28,000; and 120,000, respectively. The target antigens of Cam1 and Cam28 could be localised in the cuticle. Inhibition ELISAs showed that target epitopes of both monoclonal antibodies induce an antibody response in onchocerciasis patients. 153 sera from Sierra Leonean patients were tested for their individual antibody levels against antigen epitopes recognised by Cam1 and Cam28. Patients within the age of 5-8 years had the highest levels of antibodies against the Cam28-epitope, whereas patients above 60 years had almost none. Amicrofilaremic patients had higher anti-Cam28 antibody levels than microfilaremic patients and there was a significant difference between groups with no chronic skin disease and those with mild or severe signs. A high percentage of patients (80.4%) recognised the Cam1-epitope, highest antibody levels being found in patients within the age group of 15 to 45 years and in microfilaremic patients. However, levels of antibodies inhibiting monoclonal antibody Cam1 could not be correlated with presence or absence of skin disease.

Immunological crossreactivity between a cloned antigen of Onchocerca volvulus and a component of the retinal pigment epithelium.

Onchocerciasis (river blindness) is a major blinding disease in Africa, Central America, and South America. Loss of vision can be due to corneal change, optic atrophy, or chorioretinal disease. It has been suggested that autoimmunological reactions resulting from crossreactivity between parasite antigens and components of eye tissues contribute to development of ocular pathology. Using sera collected from onchocerciasis patients as a screening reagent, a cDNA clone (Ov39) has been isolated from a lambda gt11 expression library of Onchocerca volvulus. This antigen exhibits immunological crossreactivity with a component of retinal pigment epithelium cells (RPE). Antiserum raised against this recombinant peptide immunoprecipitates a 22,000 Mr antigen of adult O. volvulus and recognizes a 44,000 Mr component of bovine RPE by Western blotting. A 44,000 Mr antigen of cultured human RPE metabolically labeled with 35S-methionine can be immunoprecipitated with the same antiserum. An antigen of the same size is recognized by a rabbit antiserum raised against whole O. volvulus extract. Immunocytochemical studies on cryostat sections of the bovine eye using the antirecombinant sera localizes this antigen to the RPE.

Partial characterization of 2-beta-mercaptoethanol-soluble surface-associated antigens of Onchocerca volvulus.

Antigens were extracted from the epicuticle/cuticle of intact female Onchocerca volvulus using 2% 2-beta-mercaptoethanol and 1% SDS. In Western blot analysis a human infection serum selected for its high antibody titre against whole worm homogenates did not recognize any component solubilized by 1% SDS. However, the same serum did bind at least 7 antigens among the material extracted with 2-beta-mercaptoethanol. These antigens have apparent molecular weights (Mr) of: 15,000, 18,000, 28,000, 78,000, 98,000, 120,000 and 200,000. In ELISA using this preparation as target antigen, 151 out of 153 human infection sera gave positive results. An Onchocerca-specific IgG1 monoclonal antibody, designated Cam1, recognized the 28,000 Mr antigen, which is the most prominent antigen detected by Western blot analysis using human infection sera. In ELISA, using material affinity-purified with Cam1 as target antigen, 149 out of 153 human infection sera gave a positive IgG response. From a cDNA library three expressing clones were isolated with a rabbit serum raised against 2-beta-mercaptoethanol solubilized material. One of these clones was recognized by the monoclonal antibody Cam1.

Isolation and in vitro translation of Onchocerca volvulus mRNA.

Adult Onchocerca volvulus recovered for excised nodules by dissection or treatment with collagenase have been used as a source of RNA for in vitro translation experiments. RNA was purified using either the hot phenol/SDS procedure or the guanidine isothiocyanate protocol. Immunoprecipitation experiments performed on in vitro products demonstrate a marked heterogeneity in responses by individed human infection sera. Further immunoprecipitation experiments demonstrate cross reactivity between O. volvulus and other filarial nematodes.

Beneficial effects of adenosine triphosphate-MgCl2 administered intravenously to rabbits subjected to haemorrhagic shock.

The beneficial effects of adenosine triphosphate (ATP)-MgCl2 administered as a bolus following fluid infusion or in combination with the infusion fluid were investigated in rabbits subjected to severe but reversible haemorrhagic shock. ATP-MgCl2 treatment led to a significant improvement of the metabolic functions of lung and liver tissue. Kidney tissue showed the same tendency, but the improvement did not reach significant levels. The release of lysosomal enzymes in vivo was retarded after treatment but not stopped. The mean arterial pressure was kept at a relatively constant level when ATP-MgCl2 was infused slowly. Administration as a bolus resulted in an immediate dramatic drop in pressure, followed by recovery and then a gradual decrease to levels which appeared to be incompatible with survival.

Beneficial effect of naloxone on in vitro tissue metabolism after hemorrhagic shock.

A rabbit model of hemorrhagic shock is used in assessing the use of naloxone as an adjunct in the treatment of hemorrhagic shock. In vitro oxidation rates of labelled glucose are used as parameters of early tissue damage. Naloxone, given as an adjunct to volume replacement, significantly improves the in vitro capabilities of lung and liver tissues, but has no effect on kidney cortex. Changes in MABP are not affected by naloxone in this rabbit model, and serum lysosomal enzyme activity is not significantly improved. It is proposed that naloxone exerts some of its beneficial effect at the cellular level.