Enhanced alloresponse to platelet transfusion due to immune dysregulation following ablative chemotherapy in mice.

Introduction: Alloimmunization is common following platelet transfusion and can result in negative outcomes for recipients such as refractoriness to subsequent transfusions and rejection of transplants. Healthy people do not receive blood transfusions, and the diseases and therapies that result in a need to transfuse have significant impacts on the immunological environment to which these alloantigens are introduced. Ablative chemotherapies are common among platelet recipients and have potent immunological effects. In this study, we modeled the impact of chemotherapy on the alloresponse to platelet transfusion. As chemotherapies are generally regarded as immunosuppressive, we hypothesized that that they would result in a diminished alloresponse. Methods: Mice were given a combination chemotherapeutic treatment of cytarabine and doxorubicin followed by transfusion of allogeneic platelets, and compared to controls given no treatment, chemotherapy alone, or transfusion alone. Alloantibody responses were measured 2 weeks after transfusion, and cellular responses and growth factors were monitored over time. Results: Contrary to our hypothesis, we found that chemotherapy led to increased alloantibody responses to allogeneic platelet transfusion. This enhanced response was antigen-specific and was associated with increased CD4+ and CD8+ T cell responses. Chemotherapy led to rapid lymphocyte depletion followed by reconstitution, non-specific activation of transitional B cells with the highest levels of activation in the least mature subsets, and increased serum levels of B cell activating factor (BAFF). Conclusion: These data suggest that ablative chemotherapy can increase the risk of alloimmunization and, if confirmed clinically, that additional measures to protect these patient populations may be warranted.

Polyinosinic: polycytidylic acid induced inflammation enhances while lipopolysaccharide diminishes alloimmunity to platelet transfusion in mice.

Introduction: Alloimmune responses against platelet antigens, which dominantly target the major histocompatibility complex (MHC), can cause adverse reactions to subsequent platelet transfusions, platelet refractoriness, or rejection of future transplants. Platelet transfusion recipients include individuals experiencing severe bacterial or viral infections, and how their underlying health modulates platelet alloimmunity is not well understood. Methods: This study investigated the effect of underlying inflammation on platelet alloimmunization by modelling viral-like inflammation with polyinosinic-polycytidylic acid (poly(I:C)) or gram-negative bacterial infection with lipopolysaccharide (LPS), hypothesizing that underlying inflammation enhances alloimmunization. Mice were pretreated with poly(I:C), LPS, or nothing, then transfused with non-leukoreduced or leukoreduced platelets. Alloantibodies and allogeneic MHC-specific B cell (allo-B cell) responses were evaluated two weeks later. Rare populations of allo-B cells were identified using MHC tetramers. Results: Relative to platelet transfusion alone, prior exposure to poly(I:C) increased the alloantibody response to allogeneic platelet transfusion whereas prior exposure to LPS diminished responses. Prior exposure to poly(I:C) had equivalent, if not moderately diminished, allo-B cell responses relative to platelet transfusion alone and exhibited more robust allo-B cell memory development. Conversely, prior exposure to LPS resulted in diminished allo-B cell frequency, activation, antigen experience, and germinal center formation and altered memory B cell responses. Discussion: In conclusion, not all inflammatory environments enhance bystander responses and prior inflammation mediated by LPS on gram-negative bacteria may in fact curtail platelet alloimmunization.

Recent progress in nucleic acid detection with CRISPR.

Recent advances in CRISPR-based biotechnologies have greatly expanded our capabilities to repurpose CRISPR for the development of molecular diagnostic systems. The key attribute that allows CRISPR to be widely utilized is its programmable and highly specific nature. In this review, we first illustrate the principle of the class 2 CRISPR nucleases for molecular diagnostics which originates from their immunologic defence systems. Next, we present the CRISPR-based schemes in the application of diagnostics with amplification-assisted or amplification-free strategies. By highlighting some of the recent advances we interpret how general bioengineering methodologies can be integrated with CRISPR. Finally, we discuss the challenges and exciting prospects for future CRISPR-based biosensing development. We hope that this review will guide the reader to systematically learn the start-of-the-art development of CRISPR-mediated nucleic acid detection and understand how to apply the CRISPR nucleases with different design concepts to more general applications in diagnostics and beyond.

Isothermal Background-Free Nucleic Acid Quantification by a One-Pot Cas13a Assay Using Droplet Microfluidics.

High sensitivity and specificity nucleic acid detection has been achieved by the Cas13a collateral effect in combination with a separate recombinase polymerase amplification (RPA). However, these emerging methods cannot provide accurate quantification of nucleic acids because the two-step assay performance may be compromised if the RPA and Cas13a reactions are simply unified in a single step. In this work, we first addressed the challenges associated with enzymatic incompatibility and the macromolecular crowding effect in the one-pot assay development, making the consolidated RPA-Cas13a assay a facile and robust diagnostic tool. Next, we found that the one-pot reaction cannot precisely quantify the targets at low concentrations. Thus, by leveraging droplet microfluidics, we converted the one-pot assay to a digital quantification format, termed Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA). Due to the droplet compartmentation, MEDICA greatly accelerates the reaction and enables relative detection in 10 min and the end-point quantification in 25 min. Moreover, MEDICA facilitates the droplet binarization for counting because of background-free signals generated by trans-cleavage reporting of Cas13a. Our clinical validation highlights that CRISPR-based isothermal assays are promising for the next generation of nucleic acid quantification methods.

Droplet digital recombinase polymerase amplification (ddRPA) reaction unlocking via picoinjection.

Recombinase polymerase amplification (RPA) has been recognized as a promising isothermal amplification method for nucleic acid detection. However, the digital format of RPA is still challenging to implement due to its MgOAc-initiated reaction feature and the inherent non-specific amplification. Here we develop a Picoinjection Aided Digital reaction unLOCKing (PADLOCK) approach utilizing droplet microfluidics to achieve droplet digital RPA (ddRPA) for absolute nucleic acid quantification. By coupling a microfluidic picoinjector with a droplet generator, the reaction initiator MgOAc is dosed into droplets containing MgOAc-deprived RPA master mix for controlled digital reaction unlocking, which completely circumvents premature amplification. The discretization of the targets to a single-molecule level in confined droplets endows absolute quantification of the copy number. Coupled with CRISPR/Cas13a sensing, the ddRPA demonstrates single molecule detection ability within 30 min with significantly enhanced signal-to-noise ratio (S/N ratio>6) and uniform fluorescence signal reporting, facilitating the precise quantification of nucleic acids. Furthermore, the utility of the PADLOCK-CRISPR assay has been validated with 22 clinical samples, which generated results in 100% concordance with qPCR. We believe the coupling of droplet microfluidic technology with digital RPA will pave the way towards ultrasensitive and precise nucleic acid quantification.

Blood donor obesity is associated with changes in red blood cell metabolism and susceptibility to hemolysis in cold storage and in response to osmotic and oxidative stress.

BACKGROUND: Obesity is a global pandemic characterized by multiple comorbidities, including cardiovascular and metabolic diseases. The aim of this study was to define the associations between blood donor body mass index (BMI) and RBC measurements of metabolic stress and hemolysis. STUDY DESIGN AND METHODS: The associations between donor BMI (<25 kg/m2 , normal weight; 25-29.9 kg/m2 , overweight; and ≥30 kg/m2 , obese) and hemolysis (storage, osmotic, and oxidative; n = 18 donors) or posttransfusion recovery (n = 14 donors) in immunodeficient mice were determined in stored leukocyte-reduced RBC units. Further evaluations were conducted using the National Heart, Lung, and Blood Institute RBC-Omics blood donor databases of hemolysis (n = 13 317) and metabolomics (n = 203). RESULTS: Evaluations in 18 donors revealed that BMI was significantly (P < 0.05) and positively associated with storage and osmotic hemolysis. A BMI of 30 kg/m2 or greater was also associated with lower posttransfusion recovery in mice 10 minutes after transfusion (P = 0.026). Multivariable linear regression analyses in RBC-Omics revealed that BMI was a significant modifier for all hemolysis measurements, explaining 4.5%, 4.2%, and 0.2% of the variance in osmotic, oxidative, and storage hemolysis, respectively. In this cohort, obesity was positively associated (P < 0.001) with plasma ferritin (inflammation marker). Metabolomic analyses on RBCs from obese donors (44.1 ± 5.1 kg/m2 ) had altered membrane lipid composition, dysregulation of antioxidant pathways (eg, increased oxidized lipids, methionine sulfoxide, and xanthine), and dysregulation of nitric oxide metabolism, as compared to RBCs from nonobese (20.5 ± 1.0 kg/m2 ) donors. CONCLUSIONS: Obesity is associated with significant changes in RBC metabolism and increased susceptibility to hemolysis under routine storage of RBC units. The impact on transfusion efficacy warrants further evaluation.

Pathogen-reduced PRP blocks T-cell activation, induces Treg cells, and promotes TGF-ß expression by cDCs and monocytes in mice.

Alloimmunization against platelet-rich plasma (PRP) transfusions can lead to complications such as platelet refractoriness or rejection of subsequent transfusions and transplants. In mice, pathogen reduction treatment of PRP with UVB light and riboflavin (UV+R) prevents alloimmunization and appears to induce partial antigen-specific tolerance to subsequent transfusions. Herein, the in vivo responses of antigen-presenting cells and T cells to transfusion with UV+R-treated allogeneic PRP were evaluated to understand the cellular immune responses leading to antigen-specific tolerance. Mice that received UV+R-treated PRP had significantly increased transforming growth factor ß (TGF-ß) expression by CD11b+ CD4+ CD11cHi conventional dendritic cells (cDCs) and CD11bHi monocytes (P < .05). While robust T-cell responses to transfusions with untreated allogeneic PRP were observed (P < .05), these were blocked by UV+R treatment. Mice given UV+R-treated PRP followed by untreated PRP showed an early significant (P < .01) enrichment in regulatory T (Treg) cells and associated TGF-ß production as well as diminished effector T-cell responses. Adoptive transfer of T-cell-enriched splenocytes from mice given UV+R-treated PRP into naive recipients led to a small but significant reduction of CD8+ T-cell responses to subsequent allogeneic transfusion. These data demonstrate that pathogen reduction with UV+R induces a tolerogenic profile by way of CD11b+ CD4+ cDCs, monocytes, and induction of Treg cells, blocking T-cell activation and reducing secondary T-cell responses to untreated platelets in vivo.

Immunodeficient mice are better for modeling the transfusion of human blood components than wild-type mice.

Animal models are vital to the study of transfusion and development of new blood products. Post-transfusion recovery of human blood components can be studied in mice, however, there is a need to identify strains that can best tolerate xenogeneic transfusions, as well as to optimize such protocols. Specifically, the importance of using immunodeficient mice, such as NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, to study human transfusion has been questioned. In this study, strains of wild-type and NSG mice were compared as hosts for human transfusions with outcomes quantified by flow cytometric analyses of CD235a+ erythrocytes, CD45+ leukocytes, and CD41+CD42b+ platelets. Complete blood counts were evaluated as well as serum cytokines by multiplexing methods. Circulating human blood cells were maintained better in NSG than in wild-type mice. Lethargy and hemoglobinuria were observed in the first hours in wild-type mice along with increased pro-inflammatory cytokines/chemokines such as monocyte chemoattractant protein-1, tumor necrosis factor α, keratinocyte-derived chemokine (KC or CXCL1), and interleukin-6, whereas NSG mice were less severely affected. Whole blood transfusion resulted in rapid sequestration and then release of human cells back into the circulation within several hours. This rebound effect diminished when only erythrocytes were transfused. Nonetheless, human erythrocytes were found in excess of mouse erythrocytes in the liver and lungs and had a shorter half-life in circulation. Variables affecting the outcomes of transfused erythrocytes were cell dose and mouse weight; recipient sex did not affect outcomes. The sensitivity and utility of this xenogeneic model were shown by measuring the effects of erythrocyte damage due to exposure to the oxidizer diamide on post-transfusion recovery. Overall, immunodeficient mice are superior models for xenotransfusion as they maintain improved post-transfusion recovery with negligible immune-associated side effects.

DOCK2 Sets the Threshold for Entry into the Virtual Memory CD8+ T Cell Compartment by Negatively Regulating Tonic TCR Triggering.

The control of cytoskeletal dynamics by dedicator of cytokinesis 2 (DOCK2), a hematopoietic cell-specific actin effector protein, has been implicated in TCR signaling and T cell migration. Biallelic mutations in Dock2 have been identified in patients with a recessive form of combined immunodeficiency with defects in T, B, and NK cell activation. Surprisingly, we show in this study that certain immune functions of CD8+ T cells are enhanced in the absence of DOCK2. Dock2-deficient mice have a pronounced expansion of their memory T cell compartment. Bone marrow chimera and adoptive transfer studies indicate that these memory T cells develop in a cell-intrinsic manner following thymic egress. Transcriptional profiling, TCR repertoire analyses, and cell surface marker expression indicate that Dock2-deficient naive CD8+ T cells directly convert into virtual memory cells without clonal effector T cell expansion. This direct conversion to memory is associated with a selective increase in TCR sensitivity to self-peptide MHC in vivo and an enhanced response to weak agonist peptides ex vivo. In contrast, the response to strong agonist peptides remains unaltered in Dock2-deficient T cells. Collectively, these findings suggest that the regulation of the actin dynamics by DOCK2 enhances the threshold for entry into the virtual memory compartment by negatively regulating tonic TCR triggering in response to weak agonists.

Pathogen reduction with riboflavin and ultraviolet light induces a quasi-apoptotic state in blood leukocytes.

BACKGROUND: Alloimmunization to platelet-rich plasma (PRP) transfusions can cause adverse reactions such as platelet refractoriness or transplant rejection. Pathogen reduction treatment with ultraviolet light and riboflavin (UV + R) of allogeneic PRP was shown to reduce allogeneic antibody responses and confer partial antigen-specific immune tolerance to subsequent transfusions in mice. Studies have shown that UV + R was effective at both rapidly killing donor white blood cells (WBCs) and reducing their ability to stimulate an allogeneic response in vitro. However, the manner in which UV + R induces WBC death and its associated role in the immune response to treated PRP is unknown. METHODS AND MATERIALS: This study evaluates whether UV + R causes WBC apoptosis by examining phosphatidylserine exposure on the plasma membrane, membrane asymmetry, caspase activity, and chromatin condensation by flow cytometry. The immunogenicity of WBCs killed with UV + R versus apoptotic or necrotic pathways was also examined in vivo. RESULTS: WBCs after UV + R exhibited early apoptotic-like characteristics including phosphatidylserine exposure on the outer leaflet of the plasma membrane and loss of membrane asymmetry, but unlike canonical apoptotic cells, caspase activity and chromatin condensation were not apparent. However, in vivo studies demonstrated, unlike untreated or necrotic WBCs, both apoptotic WBCs and UV + R-treated WBCs failed to prime alloantibody responses to subsequent untreated transfusions. CONCLUSION: Overall, the mechanism of WBC death following UV + R treatment shares some membrane characteristics of early apoptosis but is distinct from classic apoptosis. Despite these differences, UV + R-treated and apoptotic WBCs both offer some protection from alloimmunization.

Allogeneic major histocompatibility complex antigens are necessary and sufficient for partial tolerance induced by transfusion of pathogen reduced platelets in mice.

BACKGROUND AND OBJECTIVES: Alloimmunization is common following transfusion with platelet-rich plasma (PRP) and can cause complications such as platelet refractoriness or transplant rejection. It has previously been shown that pathogen reduction of PRP with riboflavin and UV light (UV+R) can protect against alloimmunization in mice and induce partial tolerance to subsequent transfusions. MATERIALS AND METHODS: Using B6 H2d congenic mice, this study evaluated the relative contributions of major histocompatibility complex (MHC) antigens and minor antigens to both the alloresponse to PRP transfusion and the partial tolerance induced by UV+R treatment. RESULTS: Both total and MHC-specific alloantibody responses were highest when both MHC and minor antigens were mismatched, with lower alloantibody responses observed with MHC mismatch alone, demonstrating that allogeneic minor antigens can enhance the response to allogeneic MHC. There was a weak, but significant alloantibody response to minor antigens only. UV+R treatment protected against both major and minor antigen alloimmunization. Both allogeneic MHC and minor antigens primed an enhanced cytokine response ex vivo, though this was weaker with minor antigens, and both responses were blocked with UV+R treatment. CONCLUSION: Allogeneic MHC is both necessary and sufficient to induce the partial tolerance associated with UV+R treatment.

A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved from flagellar components.

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

Evidence-based toxicity evaluation and scheduling of Chinese herbal medicines.

ETHNOPHARMACOLOGICAL RELEVANCE: While there is an increasing number of toxicity report cases and toxicological studies on Chinese herbal medicines, the guidelines for toxicity evaluation and scheduling of Chinese herbal medicines are lacking. AIM: The aim of this study was to review the current literature on potentially toxic Chinese herbal medicines, and to develop a scheduling platform which will inform an evidence-based regulatory framework for these medicines in the community. MATERIALS AND METHODS: The Australian and Chinese regulations were used as a starting point to compile a list of potentially toxic herbs. Systematic literature searches of botanical and pharmaceutical Latin name, English and Chinese names and suspected toxic chemicals were conducted on Medline, PubMed and Chinese CNKI databases. RESULTS: Seventy-four Chinese herbal medicines were identified and five of them were selected for detailed study. Preclinical and clinical data were summarised at six levels. Based on the evaluation criteria, which included risk-benefit analysis, severity of toxic effects and clinical and preclinical data, four regulatory classes were proposed: Prohibited for medicinal usage, which are those with high toxicity and can lead to injury or death, e.g., aristolochia; Restricted for medicinal usage, e.g., aconite, asarum, and ephedra; Required warning label, e.g., coltsfoot; and Over-the-counter herbs for those herbs with a safe toxicity profile. CONCLUSION: Chinese herbal medicines should be scheduled based on a set of evaluation criteria, to ensure their safe use and to satisfy the need for access to the herbs. The current Chinese and Australian regulation of Chinese herbal medicines should be updated to restrict the access of some potentially toxic herbs to Chinese medicine practitioners who are qualified through registration.

Prevalence and predictors of traditional medicine utilization among persons living with AIDS (PLWA) on antiretroviral (ARV) and prophylaxis treatment in both rural and urban areas in South Africa.

Previous studies have reported that majority of antiretroviral (ARV) treatment-naïve patients use traditional medicine (TM). Given that TM use is ubiquitous in South Africa especially for chronic conditions, there is a potential for ARV non-adherence and serious drug interactions among patients with HIV/AIDs who use TM. The motivating factors for TM use in HIV/AIDS patients on ARV and prophylaxis treatment have not been well defined in South Africa. This study aimed to investigate the prevalence, facilitators, predictors, and types of TM used among persons living with HIV/AIDS on antiretroviral treatment. The study was a cross-sectional survey which involved 100 participants enrolled at ARV clinics in two South African provinces. Univariate and bivariate analyses were performed to assess the relationships between variables and potential predictors of TM. Sixteen percent of participants on ARV reported TM use. Seventy-nine percent used TM prior to a diagnosis of HIV. Participants were more likely to use TM if they were from a rural province, female, older, unmarried, employed, had limited education, or were HIV-positive for less than five years. TM users reported utilizing herbal or medicinal mixtures that were claimed to heal all conditions. This study provides insights into the treatment modalities selected by patients with HIV/AIDS in South Africa who are receiving ARV. This study revealed that less than 20% of participants co-used TM and ARV. However, close to 80% of participants utilize TM before contracting HIV, which is in keeping with approximate estimates by the WHO.

Why HIV positive patients on antiretroviral treatment and/or cotrimoxazole prophylaxis use traditional medicine: perceptions of health workers, traditional healers and patients: a study in two provinces of South Africa.

The study explored the perceptions, knowledge and attitudes of patients, health workers and traditional healers about the use of traditional medicine and Anti Retroviral Therapy (ART). The study explored the perceptions, knowledge and attitudes of patients, health workers and traditional healers about the use of traditional medicine and Anti Retroviral Therapy (ART), using an exploratory qualitative design in two provinces of South Africa: an urban township health facility in the Western Cape, and a rural district hospital in KwaZulu-Natal (KZN) with antennal HIV rate of 32% and 28%'respectively. In-depth interviews were conducted with 14 participants: six HIV patients on ART and using Traditional Medicine(TM), two doctors, two nurses and four traditional healers. Two focus group discussions -one at each site - were held with community health workers who work with HIV-positive patients (Western Cape [5] and in KZN [4]). Patient said to have used Traditional Healing Practices (THP) before they were diagnosed with HIV, and some who have been diagnosed with HIV continue using TM in conjunction with ART and/or Cotrimoxazole prophylaxis. Patients preferred not to disclose THP to health professionals because of lack of support and understanding. Patients utilize THP because of family expectations, privacy and confidentiality, especially when they have not disclosed their HIV status. Healthcare professionals had strong negative opinions about THP, especially for HIV-positive patients. Traditional healers supported the patient's rationale for THP use. This study revealed a need to better understand factors involved in patients' choosing to use THP concurrently with ART.

SPM1 stabilizes subpellicular microtubules in Toxoplasma gondii.

We have identified two novel proteins that colocalize with the subpellicular microtubules in the protozoan parasite Toxoplasma gondii and named these proteins SPM1 and SPM2. These proteins have basic isoelectric points and both have homologs in other apicomplexan parasites. SPM1 contains six tandem copies of a 32-amino-acid repeat, whereas SPM2 lacks defined protein signatures. Alignment of Toxoplasma SPM2 with apparent Plasmodium SPM2 homologs indicates that the greatest degree of conservation lies in the carboxy-terminal half of the protein. Analysis of Plasmodium homologs of SPM1 indicates that while the central 32-amino-acid repeats have expanded to different degrees (7, 8, 9, 12, or 13 repeats), the amino- and carboxy-terminal regions remain conserved. In contrast, although the Cryptosporidium SPM1 homolog has a conserved carboxy tail, the five repeats are considerably diverged, and it has a smaller amino-terminal domain. SPM1 is localized along the full length of the subpellicular microtubules but does not associate with the conoid or spindle microtubules. SPM2 has a restricted localization along the middle region of the subpellicular microtubules. Domain deletion analysis indicates that four or more copies of the SPM1 repeat are required for localization to microtubules, and the amino-terminal 63 residues of SPM2 are required for localization to the subpellicular microtubules. Gene deletion studies indicate that neither SPM1 nor SPM2 is essential for tachyzoite viability. However, loss of SPM1 decreases overall parasite fitness and eliminates the stability of subpellicular microtubules to detergent extraction.

RNG1 is a late marker of the apical polar ring in Toxoplasma gondii.

The asexually proliferating stages of apicomplexan parasites cause acute symptoms of diseases such as malaria, cryptosporidiosis and toxoplasmosis. These stages are characterized by the presence of two independent microtubule organizing centers (MTOCs). Centrioles are found at the poles of the intranuclear spindle. The apical polar ring (APR), a MTOC unique to apicomplexans, organizes subpellicular microtubules which impose cell shape and apical polarity on these protozoa. Here we describe the characteristics of a novel protein that localizes to the APR of Toxoplasma gondii which we have named ring-1 (RNG1). There are related RNG1 proteins in Neospora caninum and Sarcocystis neurona but no obvious homologs in Plasmodium spp., Cryptosporidium spp. or Babesia spp. RNG1 is a small, low-complexity, detergent-insoluble protein that assembles at the APR very late in the process of daughter parasite replication. We were unable to knock-out the RNG1 gene, suggesting that its gene product is essential. Tagged RNG1 lines have also allowed us to visualize the APR during growth of Toxoplasma in the microtubule-disrupting drug oryzalin. Oryzalin inhibits nuclear division and cytokinesis although Toxoplasma growth continues, and similar to earlier observations of unchecked centriole duplication in oryzalin-treated parasites, the APR continues to duplicate during aberrant parasite growth.

Tulbaghia alliacea phytotherapy: a potential anti-infective remedy for candidiasis.

The reproductive health of individuals is severely compromised by HIV infection, with candidiasis being the most prevalent oral complication in patients. Although not usually associated with severe morbidity, oropharyngeal candidiasis can be clinically significant, as it can interfere with the administration of medications and adequate nutritional intake, and may spread to the esophagus. Azole antifungal agents are commonly prescribed for the treatment and prophylaxis of candidal infections, however, the emergence of drug resistant strains and dose limiting toxic effects has complicated the treatment of candidiasis. Consequently, safe and effective and affordable medicine is required to combat this fungus. Commercial garlic (Allium sativum) has been used since time immemorial as a natural antibiotic, however, very little is known about the antifungal properties of two indigenous South African species of garlic, namely Tulbaghia alliacea and Tulbaghia violacea, used as folk medicines for a variety of infections. This study compares the in vitro anticandidal activity of Tulbaghia alliacea, Tulbaghia violacea and Allium sativum extracts. It was found that the greatest concentrations of inhibitory components were extracted by chloroform or water. The IC50 concentrations of Tulbaghia alliacea were 0.007-0.038% (w/v). Assays using S. cerevisiae revealed that the T. alliacea extract was fungicidal, with a killing half-life of approximately 2 h. This inhibitory effect of the T. alliacea extracts was observed via TLC, and may be due to an active compound called marasmicin, that was identified using NMR. This investigation confirms that extracts of T. alliacea exhibit anti-infective activity against candida species in vitro.

An assessment of two Carpobrotus species extracts as potential antimicrobial agents.

For centuries, indigenous people in South Africa have used a variety of medicinal herbs to treat chronic infections. This investigation focused on two Carpobrotus species belonging to the family, Aizoaceae, in an attempt to assess their antimicrobial potential. Extracts of varying polarities of the plants were prepared and tested against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Mycobacterium smegmatis. For the disc diffusion method, Ciprofloxacin (40 microg/disc) served as positive control for S. aureus, P. aeruginosa and M. smegmatis, whereas amphotericin B (25 microg/disc) was the control for C. albicans. A sample concentration of 10 mg/ml was used. Minimum inhibitory concentrations (MIC) were determined by two-fold serial dilution. Phytochemical analysis was completed to test for the presence of flavonoids, hydrolysable tannins, phytosterols and aromatic acids. The ethyl acetate extracts (21 microl of 95 mg/ml) were used for bio-autography, together with TLC analyses. Carpobrotus muirii and Carpobrotus quadrifidus showed antimicrobial activity against S. aureus and M. smegmatis in the disc diffusion method and inhibition against S. aureus and M. smegmatis was observed by clear zones on the TLC plate. This investigation confirms that extracts of these Carpobrotus species that are used as indigenous medicines, exhibit anti-bacterial activity. This scientific information can serve as an important platform for the development of inexpensive, safe and effective natural anti-infective medicines.