Prakasha Kempaiah

Mechanisms of erythropoiesis inhibition by malarial pigment and malaria-induced proinflammatory mediators in an in vitro model

One of the commonest complications of Plasmodium falciparum malaria is the development of severe malarial anemia (SMA), which is, at least in part, due to malaria‐induced suppression of erythropoiesis. Factors associated with suppression of erythropoiesis and development of SMA include accumulation of malarial pigment (hemozoin, PfHz) in bone marrow and altered production of inflammatory mediators, such as tumor necrosis factor (TNF)‐α, and nitric oxide (NO). However, studies investigating the specific mechanisms responsible for inhibition of red blood cell development have been hampered by difficulties in obtaining bone marrow aspirates from infants and young children, and the lack of reliable models for examining erythroid development. As such, an in vitro model of erythropoiesis was developed using CD34+ stem cells derived from peripheral blood to examine the effects of PfHz, PfHz‐stimulated peripheral blood mononuclear cell (PBMC)‐conditioned media (CM‐PfHz), TNF‐α, and NO on erythroid cell development. PfHz only slightly suppressed erythroid cell proliferation and maturation marked by decreased expression of glycophorin A (GPA). On the other hand, CM‐PfHz, TNF‐α, and NO significantly inhibited erythroid cell proliferation. Furthermore, decreased proliferation in cells treated with CM‐PfHz and NO was accompanied by increased apoptosis of erythropoietin‐stimulated CD34+ cells. In addition, NO significantly inhibited erythroid cell maturation, whereas TNF‐α did not appear to be detrimental to maturation. Collectively, our results demonstrate that PfHz suppresses erythropoiesis by acting both directly on erythroid cells, and indirectly via inflammatory mediators produced from PfHz‐stimulated PBMC, including TNF‐α and NO.Am. J. Hematol. 86:155–162, 2011.

Natural infection of Plasmodium brasilianum in humans: Man and monkey share quartan malaria parasites in the Venezuelan Amazon

Background The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. Methods We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. Findings Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. Interpretation This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts.

Mycobacterium abscessus Glycopeptidolipid Prevents Respiratory Epithelial TLR2 Signaling as Measured by HβD2 Gene Expression and IL-8 Release

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with “masking” of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.

Polymorphic variability in the 3' untranslated region (UTR) of IL12B is associated with susceptibility to severe anaemia in Kenyan children with acute Plasmodium falciparum malaria.

BackgroundPlasmodium falciparum malaria remains a leading cause of morbidity and mortality among African children. Innate immunity provides the first line of defence against P. falciparum infections, particularly in young children that lack naturally-acquired malarial immunity, such as the population examined here. Consistent with the fact that elevated interleukin (IL)-12 is an important component of the innate immune response that provides protective immunity against malaria, we have previously shown that suppression of IL-12 in African children is associated with the development of severe malarial anaemia (SMA). Since the role of IL12B variants in conditioning susceptibility to SMA remains largely unexplored, the association between a single nucleotide polymorphism (1188A→C, rs3212227), SMA (Hb<6.0g/dL), circulating IL-12p40/p70 levels, and longitudinal clinical outcomes in Kenyan children (n = 756) residing in a holoendemic falciparum malaria transmission area were investigated.ResultsMultivariate logistic regression analysis in children with acute malaria (n = 544) demonstrated that carriers of the C allele had increased susceptibility to SMA (CC: OR, 1.674; 95% CI, 1.006-2.673; P = 0.047, and AC: OR, 1.410; 95% CI, 0.953-2.087; P = 0.086) relative to wild type (AA). Although children with SMA had lower IL-12p40/p70 levels than the non-SMA group (P = 0.037), levels did not differ significantly according to genotype. Longitudinal analyses in the entire cohort (n = 756) failed to show any significant relationships between rs3212227 genotypes and either susceptibility to SMA or all-cause mortality throughout the three year follow-up.ConclusionThe rs3212227 is a marker of susceptibility to SMA in children with acute disease, but does not appear to mediate functional changes in IL-12 production or longitudinal outcomes during the acquisition of naturally-acquired malarial immunity.

Functional Promoter Haplotypes of Interleukin-18 Condition Susceptibility to Severe Malarial Anemia and Childhood Mortality

ABSTRACT Severe malarial anemia (SMA) is a leading cause of morbidity and mortality in children residing in regions where Plasmodium falciparum transmission is holoendemic. Although largely unexplored in children with SMA, interleukin-18 (IL-18) is important for regulating innate and acquired immunity in inflammatory and infectious diseases. As such, we selected two functional single-nucleotide polymorphisms (SNPs) in the IL-18 promoter (−137G→C [rs187238] and −607C→A [rs1946518]) whose haplotypes encompass significant genetic variation due to the presence of strong linkage disequilibrium among these variants. The relationship between the genotypes/haplotypes, SMA (hemoglobin [Hb], <5.0 g/dl], and longitudinal clinical outcomes were then investigated in Kenyan children (n = 719). Multivariate logistic regression analyses controlling for age, gender, sickle cell trait, glucose-6-phosphate dehydrogenase (G6PD) deficiency, HIV-1, and bacteremia revealed that carriage of the −607AA genotype was associated with protection against SMA (odds ratio [OR] = 0.440 [95% confidence interval {CI} = 0.21 to 0.90], P = 0.031) in children with acute infection. In contrast, carriers of the −137G/−607C (GC) haplotype had increased susceptibility to SMA (OR = 2.050 [95% CI = 1.04 to 4.05], P = 0.039). Measurement of IL-18 gene expression in peripheral blood leukocytes demonstrated that elevated IL-18 transcripts were associated with reduced hemoglobin concentrations (ρ = −0.293, P = 0.010) and that carriers of the “susceptible” GC haplotype had elevated IL-18 transcripts (P = 0.026). Longitudinal investigation of clinical outcomes over a 3-year follow-up period revealed that carriers of the rare CC haplotype (∼1% frequency) had 5.76 times higher mortality than noncarriers (P = 0.001). Results presented here demonstrate that IL-18 promoter haplotypes that condition elevated IL-18 gene products during acute infection are associated with increased risk of SMA. Furthermore, carriage of the rare CC haplotype significantly increases the risk of childhood mortality.

Human tissue factor pathway inhibitor-2 is internalized by cells and translocated to the nucleus by the importin system.

Tissue factor pathway inhibitor-2 (TFPI-2) is a serine proteinase inhibitor that induces caspase-mediated apoptosis when offered to a variety of tumor cells. In order to investigate the mechanism of TFPI-2-induced apoptosis, we initially studied the uptake and trafficking of TFPI-2 by HT-1080 cells. Exogenously offered TFPI-2 was rapidly internalized and distributed in both the cytosolic and nuclear fractions. Nuclear localization of TFPI-2 was also detected in a variety of endothelial cells constitutively expressing TFPI-2. Nuclear localization of TFPI-2 required a NLS sequence located in its Lys/Arg-rich C-terminal tail comprising residues 191-211, as a TFPI-2 construct lacking the C-terminal tail failed to localize to the nucleus. Complexes of TFPI-2 and importin-alpha were co-immunoprecipitated from cell lysates of HT-1080 cells either offered or overexpressing this protein, providing evidence that TFPI-2 was shuttled to the nucleus by the importin system. Our results provide the initial description of TFPI-2 internalization and translocation to the nucleus in a number of cells.

Reduced systemic bicyclo‐prostaglandin‐E2 and cyclooxygenase‐2 gene expression are associated with inefficient erythropoiesis and enhanced uptake of monocytic hemozoin in children with severe malarial anemia

In holoendemic Plasmodium falciparum transmission areas, severe malaria primarily occurs in children aged <48 months and manifests as severe malarial anemia [SMA; hemoglobin (Hb) < 6.0 g/dL]. Induction of high levels of prostaglandin‐E2 (PGE2) through inducible cyclooxygenase‐2 (COX‐2) is an important host‐defense mechanism against invading pathogens. We have previously shown that COX‐2‐derived PGE2 levels are reduced in children residing in hyperendemic transmission regions with cerebral malaria and in those with mixed sequelae of anemia and hyperparasitemia. Our in vitro studies further demonstrated that reduced PGE2 was due to downregulation of COX‐2 gene products following phagocytosis of malarial pigment (hemozoin, PfHz). However, as COX‐2‐PGE2 pathways and the impact of naturally acquired PfHz on erythropoietic responses have not been determined in children with SMA, plasma and urinary bicyclo‐PGE2/creatinine and leukocytic COX‐2 transcripts were determined in parasitized children (<36 months) stratified into SMA (n = 36) and non‐SMA (Hb ≥ 6.0 g/dL; n = 38). Children with SMA had significantly reduced plasma (P = 0.001) and urinary (P < 0.001) bicyclo‐PGE2/creatinine and COX‐2 transcripts (P = 0.007). There was a significant positive association between Hb and both plasma (r = 0.363, P = 0.002) and urinary (r = 0.500, P = 0.001)] bicyclo‐PGE2/creatinine. Furthermore, decreased systemic bicyclo‐PGE2/creatinine was associated with inefficient erythropoiesis (i.e., reticulocyte production index; RPI < 2.0, P = 0.026). Additional analyses demonstrated that plasma (P = 0.031) and urinary (P = 0.070) bicyclo‐PGE2/creatinine and COX‐2 transcripts (P = 0.026) progressively declined with increasing concentrations of naturally acquired PfHz by monocytes. Results presented here support a model in which reduced COX‐2‐derived PGE2, driven in part by naturally acquired PfHz by monocytes, promotes decreased erythropoietic responses in children with SMA. Am. J. Hematol., 2012.

Comparative Effects of Aprotinin and Human Recombinant R24K KD1 on Temporal Renal Function in Long-Evans Rats

Bovine aprotinin, a reversible inhibitor of plasmin and kallikrein, has been clinically approved for over two decades to prevent perioperative blood loss during cardiac surgery. However, because of postoperative renal dysfunction in thousands of these patients, aprotinin was voluntarily withdrawn from the market. Our earlier studies indicated that a R24K mutant of the first Kunitz-type domain of human tissue factor pathway inhibitor-2 (R24K KD1) exhibited plasmin inhibitory activity equivalent to aprotinin in vitro. In this study, we compared the effects on renal function after infusion of aprotinin and recombinant R24K KD1 in chronically instrumented, conscious rats. Aprotinin-infused rats exhibited statistically significant decreases in glomerular filtration rate and effective renal plasma flow relative to rats infused with phosphate-buffered saline (PBS) or R24K KD1 dissolved in PBS. In addition, aprotinin-treated rats exhibited marked increases in serum creatinine, blood urea nitrogen, urinary protein, and effective renal vascular resistance, whereas these renal parameters remained essentially unchanged in vehicle and R24K KD1-treated rats for a one-week period. Moreover, with use of a highly sensitive apoptosis detection assay, a significant increase in the rate of early and late apoptotic events in renal tubule cells occurred in aprotinin-treated rats relative to R24K KD1-treated rats. In addition, histological examination of the rat kidney revealed markedly higher levels of protein reabsorption droplets in the aprotinin-infused rats. Our data collectively provide suggestive evidence that R24K KD1 does not induce the renal dysfunction associated with aprotinin, and may be an effective clinical alternative to aprotinin as an antifibrinolytic agent in cardiac surgery.

Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation

Bacteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[−]) enteric Bacilli and Plasmodium falciparum (Pf[+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/μL), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children (n = 206, aged <3 yrs): healthy; Pf[+] alone; G[−] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[−] organisms, respectively. Coinfected children, particularly those with G[−] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN-γ, and IFN-α and decreased TNF-α relative to malaria alone. Children with G[−] coinfection had higher IL-1β and IL-1Ra and lower IL-10 than the Pf[+] group and higher IFN-γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN-γ. Results here suggest that enhanced immune activation, especially in G[−] coinfected children, acts to reduce malaria parasite burden.

Cystic fibrosis CFBE41o‐ cells contain TLR1 SNP I602S and fail to respond to Mycobacterium abscessus

BACKGROUND Mycobacterium abscessus causes lung infection in patients with cystic fibrosis. M. abscessus stimulates the host innate immune response via TLR2 on respiratory epithelial cells. Signaling through TLR2 requires the formation of TLR2/TLR1 heterodimers on the cell surface. METHODS The ability of M. abscessus to stimulate the innate immune response of cystic fibrosis CFBE41o- respiratory epithelial cells was measured as expression of HβD2 by RT PCR, and release of IL-8 by ELISA. Genotyping of CFBE41o- TLR polymorphisms was carried out. RESULTS CFBE41o- cells are hyporesponsive to M. abscessus. They are homozygous for the TLR1 SNP I602S which has been demonstrated to cause diminished cellular responses to TLR2 agonists. CONCLUSIONS Homozygosity for I602S is prevalent in Western Europeans and North American Caucasians, the same demographic in which the ΔF508 mutation is present. This SNP may play a role in the pathogenesis of M. abscessus lung infection in patients with cystic fibrosis.