Direct contact between Plasmodium falciparum and human B-cells in a novel co-culture increases parasite growth and affects B-cell growth.

BACKGROUND: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies. METHODS AND RESULTS: A new method for growing P. falciparum in 5% CO2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 - FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed. CONCLUSIONS: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

Factors influencing the induction of high affinity antibodies to Plasmodium falciparum merozoite antigens and how affinity changes over time.

Understanding the functional characteristics of naturally acquired antibodies against P. falciparum merozoite antigens is crucial for determining the protective functions of antibodies. Affinity (measured as kd) of naturally acquired antibodies against two key targets of acquired immunity, EBA175 and PfRh2, was determined using Surface Plasmon Resonance (SPR) in a longitudinal survey in Nigeria. A majority of the participants, 79% and 67%, maintained stable antibody affinities to EBA175 and PfRh2, respectively, over time. In about 10% of the individuals, there was a reciprocal interaction with a reduction over time in antibody affinity for PfRh2 and an increase for EBA175. In general, PfRh2 elicited antibodies with higher affinity compared to EBA175. Individuals with higher exposure to malaria produced antibodies with higher affinity to both antigens. Younger individuals (5-15 years) produced comparable or higher affinity antibodies than adults (>15 years) against EBA175, but not for PfRh2. Correlation between total IgG (ELISA) and affinity varied between individuals, but PfRh2 elicited antibodies with a higher correlation in a majority of the participants. There was also a correlation between antibody inhibition of erythrocyte invasion by merozoites and PfRh2 affinity. This work gives new insights into the generation and maintenance of antibody affinity over time.

Development of Plasmodium falciparum specific naïve, atypical, memory and plasma B cells during infancy and in adults in an endemic area.

BACKGROUND: B-cells are essential in immunity against malaria, but which sub-sets of B-cells specifically recognize Plasmodium falciparum and when they appear is still largely unknown. RESULTS: Using the flow cytometry technique for detection of P. falciparum specific (Pf+) B-cells, this study for the first time measured the development of Pf+ B cell (CD19+) phenotypes in Ugandan babies from birth up to nine months, and in their mothers. The babies showed increases in Pf+ IgG memory B-cells (MBCs), atypical MBCs, and plasma cells/blasts over time, but the proportion of these cells were still lower than in the mothers who displayed stable levels (5, 18, and 3%, respectively). Pf+ non-IgG+ MBCs and naïve B-cells binding to P. falciparum antigens were higher in the babies compared to the mothers (12 and 50%). In ELISA there was an increase in IgG and IgM antibodies over time in babies, and stable levels in mothers. At baby delivery, multigravidae mothers had a higher proportion of Pf+ IgG MBCs and less Pf+ naïve B-cells than primigravidae mothers. CONCLUSIONS: In newborns, naïve B-cells are a major player in recognizing P. falciparum. In adults, the high proportion of Pf+ atypical MBCs suggests a major role for these cells. Both in infants and adults, non-IgG+ MBCs were higher than IgG MBCs, indicating that these cells deserve more focus in future.

Novel flow cytometry technique for detection of Plasmodium falciparum specific B-cells in humans: increased levels of specific B-cells in ongoing infection.

BACKGROUND: Malaria caused by Plasmodium falciparum is still a major health threat in endemic areas especially for children below 5 years of age. While it is recognized that antibody immunity plays an important role in controlling the disease, knowledge of the mechanisms of sustenance and natural boosting of immunity is very limited. Before, it has not been possible to investigate malaria specific B-cells directly in flow cytometry, making it difficult to know how much of a B cell response is due to malaria, or how much is due to other immunological stimulators. METHODS: This study developed a technique using quantum dots and schizont extract made from ghosts of infected erythrocytes, to be able to investigate P. falciparum specific B-cells, something that has never been done before. RESULTS: Major differences in P. falciparum specific B-cells were found between samples from immune (22.3 %) and non-immune (1.7 %) individuals. Samples from parasite positive individuals had the highest proportions of specific B-cells (27.9 %). CONCLUSION: The study showed increased levels of P. falciparum-specific B-cells in immune individuals, with the highest levels in active malaria infections, using a new technique that opens up new possibilities to study how these cells are sustained in vivo after natural infections. It will also be useful in vaccine studies.

Differences in affinity of monoclonal and naturally acquired polyclonal antibodies against Plasmodium falciparum merozoite antigens.

BACKGROUND: Malaria is a major global cause of deaths and a vaccine is urgently needed. RESULTS: We have employed the P. falciparum merozoite antigens MSP2-3D7/FC27 and AMA1, used them in ELISA, and coupled them in different ways using surface plasmon resonance (SPR) and estimated affinity (measured as kd) of monoclonal as well as naturally-acquired polyclonal antibodies in human plasma. There were major differences in kd depending on how the antigens were immobilized and where the His-tag was placed. For AMA1 we could see correlations with invasion inhibition. Using different immobilizations of proteins in SPR, we could see only moderate correlations with levels of antibodies in ELISA, indicating that in ELISA the proteins were not uniformly bound and that antibodies with many specificities exist in natural immunisation. The correlations between ELISA and SPR were enhanced when only parasite positive samples were included, which may indicate that high affinity antibodies are difficult to maintain over long periods of time. We found higher kd values for MSP2 (indicating lower affinity) compared to AMA1, which might be partly explained by MSP2 being an intrinsically disordered protein, while AMA1 is globular. CONCLUSIONS: For future vaccine studies and for understanding immunity, it is important to consider how to present proteins to the immune system to achieve highest antibody affinities.

High affinity antibodies to Plasmodium falciparum merozoite antigens are associated with protection from malaria.

BACKGROUND: Malaria kills almost 1 million people every year, but the mechanisms behind protective immunity against the disease are still largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, surface plasmon resonance technology was used to evaluate the affinity (measured as k(d)) of naturally acquired antibodies to the Plasmodium falciparum antigens MSP2 and AMA1. Antibodies in serum samples from residents in endemic areas bound with higher affinities to AMA1 than to MSP2, and with higher affinities to the 3D7 allele of MSP2-3D7 than to the FC27 allele. The affinities against AMA1 and MSP2-3D7 increased with age, and were usually within similar range as the affinities for the monoclonal antibodies also examined in this study. The finding of MSP2-3D7 type parasites in the blood was associated with a tendency for higher affinity antibodies to both forms of MSP2 and AMA1, but this was significant only when analyzing antibodies against MSP2-FC27, and individuals infected with both allelic forms of MSP2 at the same time showed the highest affinities. Individuals with the highest antibody affinities for MSP2-3D7 at baseline had a prolonged time to clinical malaria during 40 weeks of follow-up, and among individuals who were parasite positive at baseline higher antibody affinities to all antigens were seen in the individuals that did not experience febrile malaria during follow up. CONCLUSIONS/SIGNIFICANCE: This study contributes important information for understanding how immunity against malaria arises. The findings suggest that antibody affinity plays an important role in protection against disease, and differs between antigens. In light of this information, antibody affinity measurements would be a key assessment in future evaluation of malaria vaccine formulations.

Acoustic wave immunosensing of a meningococcal antigen using gold nanoparticle-enhanced mass sensitivity.

Bacterial meningitis is an infection of the thin membranes covering the brain and spinal cord by a number of microorganisms including Neisseria meningitidis, which can lead to permanent neurological damage in the event of late diagnosis. Given the quick onset and severity of the disease, there is a clear need for a rapid, sensitive and specific diagnostic technique. Here, we describe the development and evaluation of an acoustic wave sensor, the quartz crystal microbalance (QCM), as a rapid immunosensor employing antibodies against the cell surface outer membrane protein 85 (OMP85) of N. meningitidis as an immobilized selective layer. These antibodies were directionally orientated as receptors by thin film deposition of structured polyvinylidene fluoride and Protein A. The sensitivity of this QCM immunosensor was further increased by conjugation of the OMP85 antigen to 50 nm gold nanoparticles providing reproducible detection of the target down to 300 ng/mL. Subsequent treatment of the QCM surface with an acidic glycine solution regenerated the immunosensor allowing each crystal to be used several times.

Histomorphometric and sympathetic innervation of the human renal artery: A cadaveric study.

BACKGROUND AND AIM: Renal artery stenosis (RAS) and acute renal failure may be due to the intimal hyperplasia and sympathetic fibers of the renal artery (RA), respectively. The purpose of this study was to characterize arterial wall and sympathetic innervation of the human RA. MATERIALS AND METHODS: Fifty-two fresh human RA samples (proximal part) were collected from 26 cadavers (19 males and 7 females), between the ages of 19 and 83 years, during autopsy. Samples were divided into three age groups: Group 1, 19-40 years; Group 2, 41-60 years; Group 3, over 61 years. 5-µm thick sections of each sample were taken and stained with hematoxylin-eosin and Verhoeff-Van Gieson. Five out of 52 samples were processed for tyrosine hydroxylase (TH) immunostaining. RESULTS: Our histological studies revealed that tunica media of RA showed smooth muscle cells and fine irregularly arranged elastic fibers. Intimal hyperplasia was the most common finding. The present study showed that thickness of tunica intima and media were found to increase with age. Sympathetic nerves were present in the tunica adventitia and outer media of the RA. The mean adventitial and sympathetic nerve fiber areas were found to be 0.595 and 0.071 mm(2), respectively. Sympathetic index (SI) to RA was calculated by dividing the sympathetic fiber area by the adventitial area of the RA. SI of RA was found to be 0.140. CONCLUSION: We conclude that RA showed the structure of musculo-elastic artery. SI may be used for the analysis of sympathetic fiber related problems of the human RA or kidneys.

Histomorphometric and sympathetic innervation of the human superficial temporal artery.

CONTEXT: Following microvascular surgeries, stenosis and spasm of the arterial graft or the recipient vessel are serious complications which are often caused by intimal hyperplasia and perivascular nerves, respectively. AIMS: The purpose of this study was to understand the characteristics of arterial wall and sympathetic innervation of the human superficial temporal artery (STA) and also, the effect of aging on STA. METHODS AND MATERIALS: Fifty-two fresh human STA (frontal branch) samples were obtained from 26 cadavers (19 males and 7 females) between the ages of 19 and 83 years. Samples were divided into three age groups: G1, 19-40 years; G2, 41-60 years; G3, over 61 years. 5µm-thin sections of each sample were taken and stained with haematoxylin-eosin, Verhoff's and tyrosine hydroxylase (TH) immunostaining. RESULTS: The well-defined internal elastic lamina (IEL) was observed in all samples of STA, whereas external elastic lamina (EEL) was not prominent in almost all cases or absent in few cases. This might be the important factor in the process of intimal and medial hyperplasia in the frontal branch of STA. Notably, intimal thickening appeared from second decade of life. Sympathetic fibres are located mainly in tunica adventitia and outer media. Mean adventitial and sympathetic areas were found to be 0.080 and 0.010mm(2), respectively. STATISTICAL ANALYSIS USED: One-way ANOVA followed by Tukey HSD post hoc test by using the SPSS 11.5 software. CONCLUSIONS: STA is prone to age related pathological changes. Sympathetic index may be used for analysis of sympathetic fibre-related problems (vasospasm, migraine) of the STA.

Histomorphometric and sympathetic innervation of the human internal thoracic artery.

INTRODUCTION: Internal thoracic artery (ITA) is an established arterial graft for the coronary artery by-pass surgery. Special micro-anatomical features of the ITA wall may protect it from age related pathological changes. One of the complications seen after coronary artery bypass grafting is vasospasm. Sympathetic nerves may be involved in vasospasm. OBJECTIVE: To ascertain the sympathetic innervation of the internal thoracic artery and to assess the effect of aging on this artery by histomorphometry. METHOD: Fifty-four human internal thoracic artery samples were collected from 27 cadavers (19 male and 8 female) with ages of 19 to 83 years. Samples were divided into three age groups: G1, 19-40 years; G2, 41-60 years; G3, >61 years. Sections (thickness 5 mm) of each sample were taken and stained with hematoxylin-eosin and Verhoeff-Van Gieson stains. Five of fifty-four samples were processed for tyrosine hydroxylase immunostaining. RESULTS: The thickness of the tunica intima was found to be constant in all age groups, whereas the thickness of the tunica media decreased in proportion to age. Verhoeff-Van Gieson staining showed numerous elastic laminae in the tunica media. Significant differences (p < 0.0001) in the number of elastic laminae were found between G1 with G2 cadavers, between G2 and G3 cadavers and between G3 and G1 cadavers. Tyrosine hydroxylase immunostaining demonstrated sympathetic fibers, located mainly in the tunica adventitia and the adventitia-media border. The sympathetic nerve fiber area and sympathetic index were found to be 0.0016 mm² and 0.012, respectively. DISCUSSION: Histology of the ITA showed features of the elastic artery. This may be associated with lower incidence of Atherosclerosis or intimal hyperplasia in ITA samples even in elderly cases. Low sympathetic index (0.012) of ITA may be associated with fewer incidences of sympathetic nervous systems problems (vasospasm) of the ITA. CONCLUSION: Sympathetic nerve fibers are present in the adventitia of the internal thoracic artery. This is an elastic artery, although anatomically it is considered to be medium-sized. The sympathetic index may be used for analysis of sympathetic nerve fiber-related problems of the internal thoracic artery.

A common trunk of origin of the right testicular and middle suprarenal arteries with a retrocaval course.

A common trunk of origin of the right testicular and middle suprarenal arteries with a retrocaval course was observed during the dissection of a male cadaver. The Common trunk (CT) arose from the anterior aspect of the abdominal aorta (AA) at the level of the right renal artery (RRA) and after a short course behind the inferior vena cava (IVC), the CT divided into right testicular and middle suprarenal arteries. The middle suprarenal artery (MSA) passed upwards behind the IVC to the right suprarenal gland. The right testicular artery (RTA) descended posterior to the RRA and anterior to the IVC. It then continued on its normal route distally with the right testicular vein. The awareness of such variations of testicular and middle suprarenal arteries and their unusual origin and course might complicate the interpretation of angiograms and surgical procedures in the posterior abdominal area.

Histomorphometric and sympathetic innervation of the human internal thoracic artery

INTRODUCTION: Internal thoracic artery (ITA) is an established arterial graft for the coronary artery by-pass surgery. Special micro-anatomical features of the ITA wall may protect it from age related pathological changes. One of the complications seen after coronary artery bypass grafting is vasospasm. Sympathetic nerves may be involved in vasospasm. OBJECTIVE: To ascertain the sympathetic innervation of the internal thoracic artery and to assess the effect of aging on this artery by histomorphometry. METHOD: Fifty-four human internal thoracic artery samples were collected from 27 cadavers (19 male and 8 female) with ages of 19 to 83 years. Samples were divided into three age groups: G1, 19-40 years; G2, 41-60 years; G3, >61 years. Sections (thickness 5 mm) of each sample were taken and stained with hematoxylin-eosin and Verhoeff-Van Gieson stains. Five of fifty-four samples were processed for tyrosine hydroxylase immunostaining. RESULTS: The thickness of the tunica intima was found to be constant in all age groups, whereas the thickness of the tunica media decreased in proportion to age. Verhoeff-Van Gieson staining showed numerous elastic laminae in the tunica media. Significant differences (p<0.0001) in the number of elastic laminae were found between G1 with G2 cadavers, between G2 and G3 cadavers and between G3 and G1 cadavers. Tyrosine hydroxylase immunostaining demonstrated sympathetic fibers, located mainly in the tunica adventitia and the adventitia-media border. The sympathetic nerve fiber area and sympathetic index were found to be 0.0016 mm² and 0.012, respectively. DISCUSSION: Histology of the ITA showed features of the elastic artery. This may be associated with lower incidence of Atherosclerosis or intimal hyperplasia in ITA samples even in elderly cases. Low sympathetic index (0.012) of ITA may be associated with fewer incidences of sympathetic nervous systems problems (vasospasm) of the ITA. CONCLUSION: Sympathetic ...

Intra-individual and intra-species heterogeneity in nuclear rDNA ITS region of Vigna species from subgenus Ceratotropis.

The extent of intra-individual and intra-species heterogeneity in the nuclear rDNA internal transcribed spacer (ITS) was investigated among the 'Asiatic Vigna' species (subgenus Ceratotropis). High intra- and inter-individual ITS polymorphism was observed among Vigna radiata accessions, where multiple ITS length variants ranging from approximately 700 to approximately 770 bp were detected on PCR amplification. Subsequent analysis revealed that the variants are 'heteroduplex ITS fragments' generated during the PCR process. Analysis of ITS from wild and cultivated forms of ten Vigna species from subgenus Ceratotropis revealed substantial intra-species divergence in four species: Vigna umbellata, Vigna trilobata, V. radiata and Vigna minima. However, no other species analysed showed intra-individual ITS heterogeneity as observed in V. radiata. The results demonstrate differential evolution of ITS sequence among wild and cultivated forms of V. radiata. Evidence indicates that intra-species hybridization and a slow 'molecular drive' are responsible for this phenomenon. Sequence analysis of 5.8S, ITS1 and ITS2 and secondary-structure analysis of ITS regions indicate that the ITS variants do not belong to pseudogenic rDNA repeat units. Further, reverse transcriptase-PCR (RT-PCR) analysis showed that rDNA repeat units harbouring certain intra-individual ITS variants were transcriptionally inactive, indicating the regulation of these loci by epigenetic gene silencing. The V. radiata ITS variants, when analysed together, did not cause any phylogenetic errors at the species level.

Bilateral variation of facial artery: a case report.

Dissection of a 65-year-old male cadaver revealed bilateral anomalous facial artery. The right facial artery taking origin from the external carotid artery did not make any loop in the submandibular region, entered the face by winding round the lower border of mandible, and terminated as the inferior labial artery. The upper part of the right side face in this case was supplied by various branches of transverse facial artery, infra orbital artery and dorsal nasal artery. The origin, course and branching pattern of the left facial artery was normal except the inferior labial artery was missing from it. The venous drainage of the face was normal on both sides. This case may provide useful information for clinical applications in different fields of oral and maxillofacial surgery.

Cyamella en el hombre, su morfología y revisión de la literatura / Cyamella in man, its morphology and review of literature

A sesamoid bone is a little bone embedded in a joint capsule or tendon. We observed a very rare sesamoid bone (cyamella) in the popliteus muscle of a 48 - year - old male cadaver. Further, the related literature is reviewed and the probable functions, morphology and clinical significance of the variation are discussed.

Un hueso sesamoideo es un pequeño hueso incrustado en una cápsula articular o tendón. Observamos un raro hueso sesamoideo (cyamella) en el músculo poplíteo de un cadáver de un hombre de 48 años de edad. Además, revisamos la literatura relacionada y las funciones probables y se discute su morfología y significación clínica.

Absence of Inferior Gluteal Artery: A Rare Observation / Ausencia de la Arteria Glútea Inferior: Una Rara Observación

The gluteal region is an important anatomical and clinical area which contains muscles and vital neurovascular bundles. They are important for their clinical and morphological reasons. In this manuscript we report a rare case of absence of inferior gluteal artery. In the same specimen the superior gluteal artery was taking origin from the anterior division of internal iliac artery. The structures normally supplied by the inferior gluteal artery were supplied by a branch coming from the superior gluteal artery. The developmental and clinical significance of the anatomical variation is discussed.

La región glútea es una importante área anatómica y clínica, la cual contiene músculos y vitales elementos neurovasculares. Estos últimos son importantes por razones clínicas y morfológicas. En este trabajo describimos un raro caso de ausencia de la arteria glútea inferior. Encontramos también que la arteria glútea superior tenía su origen en la división anterior de la arteria iliaca interna. Las estructuras irrigadas normalmente por la arteria glútea inferior estaban irrigadas por una rama que se originaba de la arteria glútea superior. Son discutidos el desarrollo y el significado clínico de esta variación anatómica.