Chloroquine attenuates hemorrhagic shock-induced suppression of Kupffer cell antigen presentation and major histocompatibility complex class II antigen expression through blockade of tumor necrosis factor and prostaglandin release

Hemorrhagic shock suppresses the ability of Kupffer cells (KC) to present antigen and express the major histocompatibility complex class II (Ia) antigen. These alterations are concomitant with an enhanced release of cytokines (tumor necrosis factor [TNF], interleukin-1 [IL-1], IL-6) and prostaglandin E2 (PGE2) by KC after hemorrhagic shock. The aim of this study was to determine whether chloroquine (CQ) administration in vivo before or after hemorrhage affects the altered cytokine and PGE2 release by KC as well as the capacity of KC to present antigen and express Ia. To study this, C3H/HeN mice were bled to and maintained at a mean arterial blood pressure of 35 mm Hg for 60 minutes, followed by fluid resuscitation. Chloroquine (10 mg/kg body weight) was injected intramuscularly 2 hours before or during resuscitation following shock. The administration of CQ led to a significant reduction in the hemorrhage-induced elevation of TNF, IL-6, and PGE2 release by KC; however, IL-1 secretion was not affected by CQ. In addition, CQ treatment abolished the hemorrhage-induced increase in circulating TNF and IL-6. These changes in cytokine and PGE2 release following CQ administration correlated with a significant enhancement of the antigen-presenting capacity of KC. No differences were observed between pretreatment and posttreatment with CQ. Our data indicate that CQ selectively inhibits the release of TNF, IL-6, and PGE2 by KC, while IL-1 secretion was unaffected. Because the reduction of these inflammatory mediators was concomitant with a significant improvement of KC capacity to present antigen and express Ia, we propose that TNF, IL-6, and PGE2 play a pivotal role in the induction of posthemorrhage immunosuppression. Furthermore, the data suggest that the suppression of KC functions occurs during or after resuscitation, because posttreatment with CQ was as effective as pretreatment. Additional studies indicated that the survival of animals after hemorrhage and sepsis was significantly increased by posttreatment of hemorrhaged mice with CQ. Thus, CQ, because of its unique ability to selectively inhibit the release of inflammatory cytokines and prostaglandins, represents a potent immunomodulating agent in the treatment of conditions associated with increased cytokine release and for decreasing the mortality from sepsis after hemorrhage.     

Double-blind test of metronidazole and tinidazole in the treatment of asymptomatic Entamoeba histolytica and Entamoeba hartmanni carriers.

One hundred and fifteen persons with asymptomatic Entamoeba histolytica or E. hartmanni infection, or both, were given metronidazole (750 mg three times daily for 5 days), tinidazole (1 g twice daily on 2 consecutive days), or a starch placebo. Three post-treatment stools were examined in the 2 weeks following initiation of treatment. Cysts of E. histolytica reappeared in the stools of 37% of 30 given metronidazole, 62% of 34 given tinidazole, and 70% of 31 given placebo. Cysts of E. hartmanni reappeared in the stools of 46% of 24 given metronidazole, 69% of 16 given tinidazole, and 90% of 10 given placebo. Rapid absorption and short duration of treatment make both drugs ineffective for the treatment of ameba carriers.     

Kupffer Cells Protect Liver Sinusoidal Endothelial Cells from Fas-Dependent Apoptosis in Sepsis by Down-Regulating gp130

Endothelial cell (EC) dysfunction is a key feature of multiple organ injury, the primary cause of fatality seen in critically ill patients. Although the development of EC dysfunction in the heart and lung is well studied in sepsis, it remains unclear in the liver. Herein, we report that liver sinusoidal ECs (LSECs; defined as CD146⁺CD45⁻) exhibit increased intercellular adhesion molecule-1 (CD54) and Fas in response to sepsis induced by cecal ligation and puncture (CLP). By using magnetically enriched LSEC (CD146⁺) populations, we show evidence of marked apoptosis, with a twofold decline in viable LSECs in CLP animals compared with sham controls. These changes and increased serum alanine aminotransferase levels were all mitigated in septic Fas^−/− and Fas ligand^−/− animals. Although we previously reported increased numbers of Fas ligand expressing CD8⁺ T lymphocytes in the septic liver, CD8⁺ T-cell deficiency did not reverse the onset of LSEC apoptosis/damage. However, Kupffer cell depletion with clodronate liposomes resulted in greater apoptosis and Fas expression after CLP and a decrease in glycoprotein 130 expression on LSECs, suggesting that STAT3 activation may protect these cells from injury. Our results document a critical role for death receptor–mediated LSEC injury and show the first evidence that Kupffer cells are essential to the viability of LSECs, which appears to be mediated through glycoprotein 130 expression in sepsis.Sepsis is the leading cause of death in critically ill patients in intensive care units and arises from the development of multiple organ injury, for which treatment options are limited.^1,2 Our laboratory studies murine polymicrobial sepsis [cecal ligation and puncture (CLP) method] and focuses on the liver to understand the pathophysiological characteristics of multiple organ injury. The liver is susceptible to sepsis-induced inflammation because escaping microbial antigens/mediators arising from the gastrointestinal tract of the septic animal pass through the liver.Under normal conditions, the liver is considered to be immune tolerant (it does not respond to microbial or food antigens often derived from the portal blood system). Liver sinusoidal endothelial cells (LSECs), an important specialized cell population, contribute to maintaining liver immune tolerance.^3–6 LSECs are the predominant population in the hepatic sinusoid and form a barrier between the hepatocytes and blood, which is separated by the space of Disse.⁴ Kupffer cells (KCs), the primary resident macrophage population of the liver, are also situated within the hepatic sinusoid and strategically sit on top of LSECs.⁴ LSECs and KCs often act as the first responders during inflammation and express classic pattern recognition receptors, such as toll-like receptor-4, along with other scavenger receptors.^3,4,7 LSECs have a high endocytotic capacity; they can absorb acetylated low-density lipoprotein and act as antigen-presenting cells (APCs). They also express major histocompatibility complex class I and II, and costimulatory markers, CD80 and CD86.^3–5Importantly, unlike vascular endothelial cells (ECs), LSECs do not have an organized basement membrane and are fenestrated.⁴ These fenestrations allow for an exchange of nutrients in the parenchyma, and cross talk with hepatocytes.⁴ In fact, it has been proposed that the cross talk between hepatocytes and LSECs in the liver, are physiologically important for liver homeostasis, organogenesis, and regeneration.^8,9 However, LSECs are difficult to isolate and study due to controversy in defining these cells.¹⁰ LSECs are also phenotypically different from vascular ECs, as LSECs are often characterized as CD146⁺CD45⁻ or CD31⁺CD45⁻ expressing cells.^11–14 Although both KCs, and LSECs express Fas, it has been proposed that LSECs may undergo Fas-mediated apoptosis, making the liver, along with the retina of the eye, one of two characterized organs that has been shown to undergo Fas-mediated apoptosis.^15–17Our previous studies have shown that there is a twofold increase in the number of Fas ligand (FasL)–expressing CD8⁺ T cells in the liver, 24 hours after CLP, and a significant increase in the number of apoptotic and/or necrotic nonleukocytic cells. Unfortunately, it still remains unclear as to what cell types are undergoing apoptosis and how this process is regulated.^18,19The endothelium acts as an initial line of defense against invading pathogens, and it regulates vascular tone and permeability. Therefore, EC dysfunction is a significant problem in sepsis. However, most of what we understand is derived from nonhepatic vascular beds and cell lines, with few studies focusing on hepatic endothelium dysfunction in septic models.^20–26 Because injured ECs detach from their basement membrane, and circulate freely throughout the bloodstream, there has been great debate as whether endothelial apoptosis can be detected in vivo during sepsis.^23,27 In this study, we have attempted to determine if LSECs undergo apoptotic cell death during sepsis and what controls this process and the degree to which these changes are associated with liver injury. We found that KCs are physiologically relevant to LSEC biological characteristics and protect LSECs and, indirectly, hepatocytes from further injury during septic inflammation.     

Plasmodium falciparum antigenic diversity: Evidence of clonal population structure

Plasmodium falciparum, the agent of malignant malaria, is one of mankind’s most severe scourges. Efforts to develop preventive vaccines or remedial drugs are handicapped by the parasite’s rapid evolution of drug resistance and protective antigens. We examine 25 DNA sequences of the gene coding for the highly polymorphic antigenic circumsporozoite protein. We observe total absence of silent nucleotide variation in the two nonrepeated regions of the gene. We propose that this absence reflects a recent origin (within several thousand years) of the world populations of P. falciparum from a single individual; the amino acid polymorphisms observed in these nonrepeat regions would result from strong natural selection. Analysis of these polymorphisms indicates that: (i) the incidence of recombination events does not increase with nucleotide distance; (ii) the strength of linkage disequilibrium between nucleotides is also independent of distance; and (iii) haplotypes in the two nonrepeat regions are correlated with one another, but not with the central repeat region they span. We propose two hypotheses: (i) variation in the highly polymorphic central repeat region arises by mitotic intragenic recombination, and (ii) the population structure of P. falciparum is clonal—a state of affairs that persists in spite of the necessary stage of physiological sexuality that the parasite must sustain in the mosquito vector to complete its life cycle.     

Evidence for clonal propagation in natural isolates of Plasmodium falciparum from Venezuela

We have analyzed 75 isolates of Plasmodium falciparum, collected in Venezuela during both the dry (November) and rainy (May-July) seasons, with a range of genetic markers including antigen genes and 14 random amplified polymorphic DNA (RAPD) primers. Thirteen P. falciparum stocks from Kenya and four other Plasmodium species are included in the analysis for comparison. Cross-hybridization shows that the 14 RAPD primers reveal 14 separate regions of the parasite's genome. The P. falciparum isolates are a monophyletic clade, significantly different from the other Plasmodium species. We identify three RAPD characters that could be useful as "tags" for rapid species identification. The Venezuelan genotypes fall into two discrete genetic subdivisions associated with either the dry or the rainy season; the isolates collected in the rainy season exhibit greater genetic diversity. There is significant linkage disequilibrium in each seasonal subsample and in the full sample. In contrast, no linkage disequilibrium is detected in the African sample. These results support the hypothesis that the population structure of P. falciparum in Venezuela, but not in Africa, is predominantly clonal. However, the impact of genetic recombination on Venezuelan P. falciparum seems higher than in parasitic species with long-term clonal evolution like Trypanosoma cruzi, the agent of Chagas' disease. The genetic structure of the Venezuelan samples is similar to that of Escherichia coli, a bacterium that propagates clonally, with occasional genetic recombination.     

A new Drosophila spliceosomal intron position is common in plants

The 25-year-old debate about the origin of introns between proponents of "introns early" and "introns late" has yielded significant advances, yet important questions remain to be ascertained. One question concerns the density of introns in the last common ancestor of the three multicellular kingdoms. Approaches to this issue thus far have relied on counts of the numbers of identical intron positions across present-day taxa on the assumption that the introns at those sites are orthologous. However, dismissing parallel intron gain for those sites may be unwarranted, because various factors can potentially constrain the site of intron insertion. Demonstrating parallel intron gain is severely handicapped, because intron sequences often evolve exceedingly fast and intron phylogenetic distributions are usually ambiguous, such that alternative loss and gain scenarios cannot be clearly distinguished. We have identified an intron position that was gained independently in animals and plants in the xanthine dehydrogenase gene. The extremely disjointed phylogenetic distribution of the intron argues strongly for separate gain rather than recurrent loss. If the observed phylogenetic pattern had resulted from recurrent loss, all observational support previously gathered for the introns-late theory of intron origins based on the phylogenetic distribution of introns would be invalidated.