Impact of oxidative stress in response to malarial infection during pregnancy: Complications, histological changes, and pregnancy outcomes.

Background and Objectives: Pregnancy malaria is a major underestimated global public health problem. To understand the involvement of oxidative stress (OS) in the pathophysiology of placental malaria, OS biomarkers, malondialdehyde (MDA), uric acid (UA), and superoxide dismutase (SOD) levels were analyzed and correlated to placental histopathological changes and pregnancy outcomes. Methods: A hospital-based study was conducted in Mangaluru, Karnataka, India, to analyze the changes in hematological parameters and the serum OS biomarker levels. Histological analysis of placenta, associated complications, and pregnancy outcomes were compared using Kruskal-Wallis test, and pairwise comparison between two groups was made by Mann-Whitney U-test. Correlations were calculated by Pearson's and Spearman's rank correlations. Results: Among 105 pregnant women, 34 were healthy controls and the infected group comprised of Plasmodium Vivax (Pv) (n = 48), Plasmodium falciparum (Pf) (n = 13), and mixed (n = 10) malaria infections. Of 71 infected cases, 67.6% had mild malaria, whereas 32.4% had severe malaria. The white blood cell and C-reactive protein levels were found to increase, whereas hemoglobin, red blood cell, and platelet levels decreased during both types of malarial infections. The MDA and UA values increased and SOD levels decreased particularly during severe Pf infections. Histological changes such as syncytial knots, syncytial ruptures, and fibrinoid necrosis were observed particularly during Pf infections and leukocyte infiltration was observed in Pv malaria. Conclusion: Evaluation of MDA, UA, and SOD levels can serve as an indicator of OS during pregnancy malaria. The OS during pregnancy may lead to complications such as severe anemia, pulmonary edema, intra uterine growth retardation, premature delivery, and low birth weight, not only during Pf but also in Pv malaria. It is important to create awareness among rural and immigrant population residing in Mangaluru and its surroundings about required preventive measures and free government-supported antenatal care services.

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.).

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1ß, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1ß, and nitric oxide.

Acquired antibody responses against merozoite surface protein-119 antigen during Plasmodium falciparum and P.vivax infections in South Indian city of Mangaluru.

Merozoite surface protein-1 (MSP-1) of malaria parasites has been extensively studied as a malaria vaccine candidate and the antibody response to this protein is an important indicator of protective immunity to malaria. Mangaluru city and its surrounding areas in southwestern India are endemic to malaria with Plasmodium vivax being the most widespread and prevalent species although P. falciparum also frequently infects. However, no information is available on the level of protective immunity in this population. In this regard, a prospective hospital-based study was performed in malarial patients to assess antibody responses against the 19-kDa C-terminal portion of P. vivax and P. falciparum MSP-1 (MSP-119). Serum samples from 51 healthy endemic controls and 267 infected individuals were collected and anti-MSP-119 antibody levels were analyzed by ELISA. The possible association between the antibody responses and morbidity parameters such as malarial anemia and thrombocytopenia was investigated. Among the 267 infected cases, 144 had P. vivax and 123 had P. falciparum infections. Significant levels of anti-MSP-119 antibody were observed both in P. vivax (123/144; 85.4%) and P. falciparum (108/123; 87.9%) infected individuals. In both type of infections, the major antibody isotypes were IgG1 and IgG3. The IgG levels were found to be increased in patients with severe anemia and thrombocytopenia. The antibody levels were also higher in infected individuals who had several previous infections, although antibodies produced during previous infections were short lived. The predominance of cytophilic anti-MSP-119 IgG1 and IgG3 antibodies suggests the possibility of a dual role of Pv MSP-119 and Pf MSP-119 during malarial immunity and pathogenesis.

Clinical features and haematological parameters among malaria patients in Mangaluru city area in the southwestern coastal region of India.

The aim of this study was to assess the clinical profile, severity and complications of patients suffering from malaria in Mangaluru, a southwestern coastal city in India. A total of 579 patients, who were treated at the District Wenlock Hospital, Mangaluru, and 168 healthy controls were recruited in this study. The clinical profile, haematological and biochemical parameters, and disease complications were assessed. The majority of patients were treated as outpatients and patients who had severe clinical conditions were admitted to the hospital for treatment and supportive care. Among the total 579 patients recruited in this study, the distribution of P. vivax, P. falciparum and mixed infections were 364 (62.9%), 150 (25.9%) and 65 (11.2%), respectively. Among these, 506 (87.4%) had mild malaria, whereas 73 (12.6%) had severe malaria. Overall, the clinical features and severity of malaria in P. vivax and mixed infection patients were comparable to P. falciparum patients, albeit with some significant differences. The clinical complications in severe malaria cases included thrombocytopenia (50.7%), metabolic acidosis (30.1%), severe anaemia (26.0%), jaundice (21.9%), hepatic dysfunction (15.1%), acute renal failure (6.8%), haematuria (8.2%), hypotension (9.6%), cerebral malaria (1.4%) and acute respiratory distress syndrome (1.4%). All the patients with severe malaria recruited in our study were successfully treated and discharged. Majority of patients had mild malaria, likely due to seeking treatment soon after experiencing symptoms and/or having preexisting immune protection. However, a significant number of patients had severe malaria and required hospital admission indicating that there is a substantial need for creating awareness among vulnerable immigrant population. Implementing effective surveillance and vector control measures in malaria hotspot locations in the city and educating people about preventive measures are likely to reduce the malaria burden in this endemic region.

Association between inflammatory cytokine levels and anemia during Plasmodium falciparum and Plasmodium vivax infections in Mangaluru: A Southwestern Coastal Region of India.

BACKGROUND AND OBJECTIVES: Dysregulated production of inflammatory cytokines might play important role in anemia during malaria infection. The objective of this study was to assess the extent of anemia due to malaria, associated complications, and inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin [IL]-6, and IL-10) across varying anemic intensity during malaria infections. MATERIALS AND METHODS: A hospital-based cross-sectional study was conducted at District Wenlock hospital in Mangaluru city. Samples from 627 patients and 168 healthy controls (HC) were analyzed for level of hemoglobin (Hb), red blood cells (RBCs), and inflammatory cytokines. The blood cell parameters and inflammatory cytokines levels across varying intensity of anemia were analyzed using Kruskal-Wallis test and pair-wise comparison between two groups were by Mann-Whitney U-test. Correlations were calculated by Pearson's and Spearman rank correlations. RESULTS: Compared to HC, Hb, and RBC levels were significantly lower in infected patients. On comparison with mild anemia patients (Hb 8-10.9 g/dL), the levels of TNF-α and IL-6 were significantly elevated, whereas IL-10 levels were lower during severe anemia (SA) (Hb <5 g/dL). In this endemic setting, we found a strong negative association between Hb levels and parasitemia, Hb and TNF-α, and positive relationship with IL-10; anemic patients also had significantly high TNF-α/IL-10 ratios. SA was associated with complications such as acute renal failure (16.0%), jaundice (16.0%), metabolic acidosis (24.0%), hypoglycemia (12.0%), hyperparasitemia (4.0%), and hepatic dysfunction (16.0%). CONCLUSIONS: Contrary to its benign reputation, Plasmodium vivax (Pv) infections can also result in severe malarial anemia (SMA) and its associated severe complications similar to Plasmodium falciparum infections. Dysregulated inflammatory cytokine responses play an important role in the pathogenesis of SMA, especially during Pv infections.

Association between Inflammatory Cytokine Levels and Thrombocytopenia during Plasmodium falciparum and P. vivax Infections in South-Western Coastal Region of India.

BACKGROUND: Thrombocytopenia is a most commonly observed complication during malaria infections. Inflammatory cytokines such as IL-1, IL-6, and IL-10 have been documented in malaria induced thrombocytopaenia. This study was aimed to understand the possible relationship between inflammatory cytokines across varying degrees of thrombocytopenia during P. vivax, P. falciparum, and mixed infections. METHODS: A hospital-based cross sectional study was conducted at District Wenlock Hospital in Mangaluru, a city situated along the south-western coastal region of Arabian Sea in India. In this study, blood samples from 627 malaria patients were analyzed for infected parasite species, clinical conditions, platelet levels, and key cytokines that are produced in response to infection; samples from 176 uninfected healthy individuals were used as controls. RESULTS: The results of our study showed a high prevalence of malarial thrombocytopenia (platelets <150 ×103/µl) in this endemic settings. About 62.7% patients had mild-to-moderate levels of thrombocytopenia and 16% patients had severe thrombocytopenia (platelets <50 × 103/µl). Upon comparison of cytokines across varying degrees of thrombocytopenia, irrespective of infecting species, the levels of TNF-α and IL-10 were significantly higher during thrombocytopenia, whereas IL-6 levels were considerably lower in severe thrombocytopenia patients suffering from P. vivax or P. falciparum infections. The severe clinical complications observed in patients with malarial thrombocytopenia included severe anemia (17.5%), acute renal failure (12.7%), jaundice (27.0%), metabolic acidosis (36.5%), spontaneous bleeding (3.2%), hypoglycemia (25.4%), hyperparasitemia (4.8%), acute respiratory distress syndrome (1.6%), pulmonary edema (19.0%), and cerebral malaria (1.6%) in various combinations. CONCLUSION: Overall, the results of our study suggest that inflammatory cytokines influence the transformation of mild forms of thrombocytopenia into severe forms during malarial infections. Further studies are needed to understand the association of inflammatory cytokine responses with severe malaria complications and thrombocytopenia.

Malaria Severity in Mangaluru City in the Southwestern Coastal Region of India.

Dakshina Kannada district in the Southwestern region of Karnataka state, India, including Mangaluru city is endemic to malaria. About 80% of malaria infections in Mangaluru and its surrounding areas are caused by Plasmodium vivax and the remainder is due to Plasmodium falciparum. Malaria-associated clinical complications significantly occur in this region. Here, we report the pathological conditions of 41 cases of fatal severe malaria, admitted to the district government hospital in Mangaluru city during January 2013 through December 2016. The results of clinical, hematological, and biochemical analyses showed that most of these severe malaria cases were associated with thrombocytopenia, anemia, metabolic acidosis, acute respiratory distress, and single or multi-organ dysfunction involving liver, kidney, and brain. Of the 41 fatal malaria cases, 24, 10, and seven patients had P. vivax, P. falciparum, and P. vivax and P. falciparum mixed infections, respectively. These data suggest that besides P. falciparum that is known to extensively cause severe and fatal malaria illnesses, P. vivax causes fatal illnesses substantially in this region, an observation that is consistent with recent findings in other regions.

Epidemiology, drug resistance, and pathophysiology of Plasmodium vivax malaria.

Malaria, caused by the protozoan parasites of the genus Plasmodium, is a major health problem in many countries of the world. Five parasite species namely, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, cause malaria in humans. Of these, P. falciparum and P. vivax are the most prevalent and account for the majority of the global malaria cases. In most areas of Africa, P. vivax infection is essentially absent because of the inherited lack of Duffy antigen receptor for chemokines on the surface of red blood cells that is involved in the parasite invasion of erythrocytes. Therefore, in Africa, most malaria infections are by P. falciparum and the highest burden of P. vivax infection is in Southeast Asia and South America. Plasmodium falciparum is the most virulent and as such, it is responsible for the majority of malarial mortality, particularly in Africa. Although, P. vivax infection has long been considered to be benign, recent studies have reported life-threatening consequences, including acute respiratory distress syndrome, cerebral malaria, multi-organ failure, dyserythropoiesis and anaemia. Despite exhibiting low parasite biomass in infected people due to parasite's specificity to infect only reticulocytes, P. vivax infection triggers higher inflammatory responses and exacerbated clinical symptoms than P. falciparum, such as fever and chills. Another characteristic feature of P. vivax infection, compared to P. falciparum infection, is persistence of the parasite as dormant liver-stage hypnozoites, causing recurrent episodes of malaria. This review article summarizes the published information on P. vivax epidemiology, drug resistance and pathophysiology.

Presence of novel triple mutations in the pvdhfr from Plasmodium vivax in Mangaluru city area in the southwestern coastal region of India.

BACKGROUND: Genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) are the targets of sulfadoxine-pyrimethamine (SP) present in artemisinin based combination therapy (ACT; artesunate + sulfadoxine pyrimethamine) for Plasmodium falciparum. Although SP is generally not used to treat vivax infection, mutations in dhfr and dhps that confer antifolate resistance in Plasmodium vivax are common; which may be attributed to its sympatric existence with P. falciparum. Current study was aimed to determine the pattern of mutations in dhfr and dhps in P. vivax isolates from Mangaluru region. METHODS: A total of 140 blood samples were collected from P. vivax-infected people attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of 140 isolates, 25 (18%) and 50 (36%) isolates were selected randomly for sequence analysis of pvdhfr and pvdhps genes respectively. Fragment of pvdhps and full length pvdhfr were amplified, sequenced and analysed for single nucleotide polymorphisms. dhps was analysed by PCR-RFLP also, to detect the two specific mutations (A383G and A553G). RESULTS: Analysis of pvdhps sequences from 50 isolates revealed single and double mutants at 38 and 46% respectively. Three non-synonymous mutations (K55R, S58R and S117N) were identified for pvdhfr. Among these, K55R was detected for the first time. CONCLUSIONS: The current study indicates that P. vivax dhps and dhfr mutant alleles are prevalent in this area, suggesting significant SP pressure.

Drug resistance genes: pvcrt-o and pvmdr-1 polymorphism in patients from malaria endemic South Western Coastal Region of India.

BACKGROUND: Malaria is highly prevalent in many parts of India and is mostly caused by the parasite species Plasmodium vivax followed by Plasmodium falciparum. Chloroquine (CQ) is the first-line treatment for blood stage P. vivax parasites, but cases of drug resistance to CQ have been reported from India. One of the surveillance strategies which is used to monitor CQ drug resistance, is the analysis of single nucleotide polymorphisms (SNPs) of the associated gene markers. Susceptibility to CQ can also be determined by copy number assessment of multidrug resistant gene (mdr-1). The current study has examined the prevalence of SNPs in P. vivax orthologs of P. falciparum chloroquine resistant and multi-drug resistant genes (pvcrt-o and pvmdr-1, respectively) and pvmdr-1 copy number variations in isolates from the highly endemic Mangaluru city near the South Western Coastal region of India. METHODS: A total of 140 blood samples were collected from P. vivax infected patients attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of these 140 samples, sequencing was carried out for 54 (38.5%) and 85 (60.7%) isolates for pvcrt-o and pvmdr-1, respectively. Single nucleotide polymorphisms (SNPs) in the pvcrt-o and pvmdr-1 genes were analysed by direct sequencing method, while copy number variations of 60 isolates (42. 8%) were determined by real time PCR. RESULTS: Out of 54 clinical isolates analysed for pvcrt-o, three (5.6%) showed K10 insertion and the rest had wild type sequence. This is the first report to show K10 insertion in P. vivax isolates from India. Further, out of 85 clinical isolates of P. vivax analysed for mutations in pvmdr-1 gene, only one isolate had wild type sequence (~ 1%) while the remaining (99%) carried mutant alleles. Seven non-synonymous mutations with two novel mutations (I946V and Y1028C) were observed. Of all the observed mutations in pvmdr-1 gene, T958M was most highly prevalent (present in 90% of samples) followed by F1076L (76%), and Y976F (7%). Amplification of pvmdr-1 gene was observed in 31.6% of the isolates, out of 60 amplified. CONCLUSION: The observed variations both in pvmdr-1 and pvcrt-o genes indicate a trend towards parasite acquiring CQ resistance in this endemic area.

Optimization of novel halophilic lipase production by Fusarium solani strain NFCCL 4084 using palm oil mill effluent.

Among different sources of lipases, fungal lipases have continued to attract a wide range of applications. Further, halophilic lipases are highly desirable for biodiesel production due to the need to mitigate environmental pollution caused as result of extensive use of fossil fuels. However, currently, the high production cost limits the industrial application of lipases. In order to address this issue, we have attempted to optimize lipase production by Fusarium solani NFCCL 4084 and using palm oil mill effluent (POME) based medium. The production was optimized using a combinatory approach of Plackett-Burman (PB) design, one factor at a time (OFAT) design and face centred central composite design (FCCCD). The variables (malt extract, (NH4)2SO4, CaCl2, MgSO4, olive oil, peptone, K2HPO4, NaNO3, Tween-80, POME and pH) were analyzed using PB design and the variables with positive contrast coefficient were found to be K2HPO4, NaNO3, Tween-80, POME and pH. The significant variables selected were further analyzed for possible optimum range by using OFAT approach and the findings revealed that K2HPO4, NaNO3, and Tween-80 as the most significant medium components, and thus were further optimized by using FCCCD. The optimum medium yielded a lipase with an activity of 7.8 U/ml, a significant 3.2-fold increase compared to un-optimized medium. The present findings revealed that POME is an alternative and suitable substrate for halophilic lipase production at low cost. Also, it is clearly evident that the combinatory approach employed here proved to be very effective in producing high activity halophilic lipases, in general.

Malaria prevalence in Mangaluru city area in the southwestern coastal region of India.

BACKGROUND: Malaria is highly prevalent in many parts of India and the Indian subcontinent. Mangaluru, a city in the southwest coastal region of Karnataka state in India, and surrounding areas are malaria endemic with 10-12 annual parasite index. Despite high endemicity, to-date, very little has been reported on the epidemiology and burden of malaria in this area. METHODS: A cross-sectional surveillance of malaria cases was performed among 900 febrile symptomatic native people (long-time residents) and immigrant labourers (temporary residents) living in Mangaluru city area. During each of dry, rainy, and end of rainy season, blood samples from a group of 300 randomly selected symptomatic people were screened for malaria infection. Data on socio-demographic, literacy, knowledge of malaria, and treatment-seeking behaviour were collected to understand the socio-demographic contributions to malaria menace in this region. RESULTS: Malaria is prevalent in Mangaluru region throughout the year and Plasmodium vivax is predominant species compared to Plasmodium falciparum. The infection frequency was found to be high during rainy season. Infections were markedly higher in males than females, and in adults aged 16-45 years than both younger and older age groups. Also, malaria incidence was high among immigrants compared to native population. In both groups, infection rate was directly correlated with their literacy level, knowledge on malaria, dwelling environment, and protective measures used. There was also a significant difference in treatment-seeking behaviour between these two groups. CONCLUSIONS: Malaria incidences in Mangaluru region are predominantly localized to certain hotspot areas within the city, where socioeconomically underprivileged and immigrant labourers are densely populated. These areas have inadequate sanitation and constant water stagnation, harbouring high vector density and contributing to high infection incidences. Additionally, people in these areas seldom practice preventive measures such as using bed nets. The high incidences of malaria in adults are due to minimal cloth wearing, and long working hours stretching to late evenings in places with high vector density. Instituting heightened preventive public measures by governments and creating awareness on using preventive protective and environmental hygienic measures through educational programmes may substantially reduce the risk of contracting infections in these areas and spreading to other areas.

Malaria Transmission Under an Unusual Circumstance Causing Death in Two Siblings.

Two school-going siblings from a family residing in a presumed malaria non-endemic locality ∼90 km from Mangalore city in southwestern India contracted Plasmodium falciparum infection. In both cases, misunderstanding of initial clinical symptoms as due to viral hepatitis resulted in progression to severe malaria before malaria treatment was initiated. Despite treatment at a tertiary hospital, the children died of cerebral malaria and multi-organ dysfunction. Active case detection in the affected locality suggested that the infection was transmitted from infected individuals who worked in nearby malaria-endemic areas and periodically visited their families. A lesson from this study is that lethal falciparum malaria can be transmitted in regions of India, believed to be non-endemic for the disease, resulting in fatal outcomes if diagnosis is missed or delayed. Implementation of effective surveillance and control measures as well as preparedness for malaria detection and diagnosis are necessary in areas that are potentially disposed to malaria transmission even though they are presumed to be non-endemic.

Subdural haematoma in Plasmodium falciparum and Plasmodium vivax mixed infection presenting multiple clinical complications.

A 40-year-old man was admitted to hospital with a 5 day history of fever, restlessness and altered sensorium. Peripheral blood smears showed a Plasmodium vivax and Plasmodium falciparum mixed infection as revealed by the presence of rings, schizonts and gametocyte forms of the parasites. The patient soon became unconscious due to subdural haematoma (SDH) associated with disseminated intravascular coagulation and thrombocytopenia. Immediate intervention with a right fronto-parieto temporal craniectomy, evacuation of the SDH and intravenous quinine administration resulted in the patient's complete recovery within 8 days of admission, and he was discharged in good clinical condition.

Dual stage synthesis and crucial role of cytoadherence-linked asexual gene 9 in the surface expression of malaria parasite var proteins.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate the adherence of parasite-infected red blood cells (IRBCs) to various host receptors. A previous study has shown that the parasite protein, cytoadherence-linked asexual gene 9 (CLAG9), is also essential for IRBC adherence. However, how CLAG9 influences this process remains unknown. In this study, we show that CLAG9 interacts with VAR2CSA, a PfEMP1 that mediates IRBC adherence to chondroitin 4-sulfate in the placenta. Importantly, our results show that the adherent parasites synthesize CLAG9 at two stages--the early ring and late trophozoite stages. Localization studies revealed that a substantial level of CLAG9 is located mainly at or in close proximity of the IRBC membrane in association with VAR2CSA. Upon treatment of IRBCs with trypsin, a significant amount of CLAG9 (≈150 kDa) was converted into ≈142-kDa polypeptide. Together these data demonstrate that a considerable amount of CLAG9 is embedded in the IRBC membrane such that at least a portion of the polypeptide at either N or C terminus is exposed on the cell surface. In parasites lacking CLAG9, VAR2CSA failed to express on the IRBC surface and was located within the parasite. Based on these findings, we propose that CLAG9 plays a critical role in the trafficking of PfEMP1s onto the IRBC surface. These results have important implications for the development of therapeutics for cerebral, placental, and other cytoadherence-associated malaria illnesses.

Circulating cytokines as biomarkers of alcohol abuse and alcoholism.

There are currently no consistent objective biochemical markers of alcohol abuse and alcoholism. Development of reliable diagnostic biomarkers that permit accurate assessment of alcohol intake and patterns of drinking is of prime importance to treatment and research fields. Diagnostic biomarker development in other diseases has demonstrated the utility of both open, systems biology, screening for biomarkers and more rational focused efforts on specific biomolecules or families of biomolecules. Long-term alcohol consumption leads to altered inflammatory cell and adaptive immune responses with associated pathologies and increased incidence of infections. This has led researchers to focus attention on identifying cytokine biomarkers in models of alcohol abuse. Alcohol is known to alter cytokine levels in plasma and a variety of tissues including lung, liver, and very importantly brain. A number of cytokine biomarker candidates have been identified, including: tumor necrosis factor-alpha, interleukin (IL)-1-alpha, IL-1-beta, IL-6, IL-8, IL-12, and monocyte chemoattractant protein-1. This is an emerging and potentially exciting avenue of research in that circulating cytokines may contribute to diagnostic biomarker panels, and a combination of multiple biomarkers may significantly increase the sensitivity and specificity of the biochemical tests aiding reliable and accurate detection of excessive alcohol intake.

Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria.

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPGs) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not 12-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria.

Binding affinity of Plasmodium falciparum-infected erythrocytes from infected placentas and laboratory selected strains to chondroitin 4-sulfate.

The adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in human placenta is mediated by chondroitin 4-sulfate (C4S). The C4S-adherent parasites selected from laboratory strains have been widely used for determining the C4S structural elements involved in IRBC binding and for the identification of parasite adhesive protein(s). However, as far as we know, the relative binding strength of the placental versus laboratory-selected parasites has not been reported. In this study, we show that IRBCs from the infected placentas bind to C4S about 3-fold higher than those selected for C4S adherence from laboratory strains. Although adherent parasites selected from several laboratory strains have comparable binding strengths, the one obtained from 3D7 parasites designated as 3D7N61 used for malaria genome sequencing, exhibits markedly lower binding strength. Furthermore, 3D7N61-CSA parasites lose most of the binding capacity by tenth generation in continuous culture.

Developmental stage- and cell cycle number-dependent changes in characteristics of Plasmodium falciparum-infected erythrocyte adherence to placental chondroitin-4-sulfate proteoglycan.

The adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin-4-sulfate (C4S). Although IRBC binding to C4S has been unequivocally established, the adherence characteristics of IRBCs at different stages of parasite development and through successive parasite generations after selection for C4S adherence are not known. Here we show that IRBCs acquire a significant capacity to bind to C4S at as early as 14 h and exhibit maximum binding at 22 to 26 h postinvasion. Surprisingly, the IRBC binding ability decreases by approximately 50% at the late trophozoite and schizont stages. The binding strength of the IRBCs also gradually decreases during successive generations after selection for C4S binding, and at the 32nd generation, the binding capacity was only approximately 31% of that of IRBCs at the 2nd generation, suggesting that IRBCs eventually lose their C4S-adherent capacity. We also tested the susceptibility of the adhesive protein(s) on the IRBC surface to trypsin treatment at different stages of parasite development. The data show that IRBCs with late trophozoites are more resistant to trypsin treatment than those containing early trophozoites, indicating that parasite proteins expressed on the IRBC surface during trophozoite maturation partially mask accessibility of adhesive protein for binding to C4S. These data provide important insights into the expression pattern of the C4S-adhesive protein(s) on the IRBC surface, emphasizing the need for understanding the regulation of genes involved in IRBC binding to C4S. Our data also define the parasite stage at which IRBCs are suitable for studying structural interactions with C4S.

The effect of substitution of the N-acetyl groups of N-acetylgalactosamine residues in chondroitin sulfate on its degradation by chondroitinase ABC.

Chondroitinase ABC is a lyase that degrades chondroitin sulfate, dermatan sulfate and hyaluronic acid into disaccharides. The purpose of this study was to determine the ability of chondroitinase ABC to degrade chondroitin sulfate in which the N-acetyl groups are substituted with different acyl groups. The bovine tracheal chondroitin sulfate A (bCSA) was N-deacetylated by hydrazinolysis, and the free amino groups derivatized into N-formyl, N-propionyl, N-butyryl, N-hexanoyl or N-benzoyl amides. Treatment of the N-acyl or N-benzoyl derivatives of bCSA with chondroitinase ABC and analysis of the products showed that the N-formyl, N-hexanoyl and N-benzoyl derivatives are completely resistant to the enzyme. In contrast, the N-propionyl or N-butyryl derivatives were degraded into disaccharides with slower kinetics compared to that of unmodified bCSA. The rate of degradation of bCSA derivatives by the enzyme was found to be in the order of N-acetyl>N-propionyl>>N-butyryl bCSA. These results have important implications for understanding the interaction of N-acetyl groups of glycosaminoglycans with chondroitinase ABC.