Paul A. Hossler

The Plasmodium falciparum Translationally Controlled Tumor Protein Homolog and Its Reaction with the Antimalarial Drug Artemisinin

Artemisinin and its derivatives are important new antimalarial drugs. When Plasmodium falciparum-infected erythrocytes are incubated with [10-3H]dihydroartemisinin, several malaria-specific proteins become labeled. One of these proteins is the P. falciparum translationally controlled tumor protein (TCTP) homolog. In vitro, dihydroartemisinin reacts covalently with recombinant TCTP in the presence of hemin. The association between drug and protein increases with increasing drug concentration, plateauing at approximately 1 drug/TCTP molecule. By Scatchard analysis, there appear to be 2 hemin binding sites on TCTP with dissociation constants of ∼18 μm. When the single cysteine moiety is blocked by pretreatment with iodoacetamide, hemin binding is not affected, whereas drug binding is reduced by two-thirds. Thus, TCTP reacts with artemisinin in situ and in vitro in the presence of hemin and appears to bind to hemin. The function of the malarial TCTP and the role of this reaction in the mechanism of action of artemisinin await elucidation.

Pneumocystis carinii Mutations Are Associated with Duration of Sulfa or Sulfone Prophylaxis Exposure in AIDS Patients

This study was conducted to determine whether Pneumocystis carinii dyhydropteroate synthase (DHPS) gene mutations in AIDS patients with P. carinii pneumonia (PCP) are affected by duration of sulfa or sulfone prophylaxis and influence response to sulfa or sulfone therapy. The P. carinii DHPS genes from 97 AIDS patients with PCP between 1991 and 1999 from 4 medical centers were amplified, using polymerase chain reaction (PCR), and sequenced. Mutations were observed in 76% of isolates from patients exposed to sulfa or sulfone prophylaxis compared with 23% of isolates from patients not exposed (P=.001). Duration of prophylaxis increased the risk of mutations (relative risk [RR] for each exposure month, 1.06; P=.02). Twenty-eight percent of patients with mutations failed sulfa or sulfone treatment; mutations increased the risk of sulfa or sulfone treatment failure (RR, 2.1; P=0.01). Thus, an increased duration of sulfa or sulfone prophylaxis increases the chance of developing a P. carinii mutation. The majority of patients with mutations respond to sulfa or sulfone therapy.

Pneumocystis carinii mutations associated with sulfa and sulfone prophylaxis failures in AIDS patients

Background:Failures of prophylaxis against Pneumocystis carinii pneumonia (PCP) in AIDS patients do occur, but no evidence for drug resistance has yet been presented. Objective:To determine whether mutations in the sulfa and sulfone drug target are associated with failure of prophylaxis using a sulfa-containing agent. Methods:Portions of the gene for P. carinii dihydropteroate synthase (DHPS), the sulfa and sulfone target, from 27 patients (20 of whom had AIDS) diagnosed with PCP between 1976 and 1997 were amplified using polymerase chain reaction and sequenced. Seven of the 27 patients (all of whom had AIDS) were receiving sulfa or sulfone drugs as prophylaxis for PCP. Results:Mutations were found at only two amino-acid positions and were significantly more common in patients who received sulfa/sulfone prophylaxis. Mutations were observed in five (71%) out of seven isolates from AIDS patients receiving sulfa/sulfone as prophylaxis compared with only two (15%) out of 13 specimens from AIDS patients who did not (P = 0.022). No mutations were seen in isolates from seven non-HIV-infected patients, none of whom were on prophylaxis. Mutations were only observed in specimens obtained in 1995–1997. Conclusions:Mutations in two amino-acid positions were significantly more common in AIDS patients with PCP who failed sulfa/sulfone prophylaxis. These amino acids appeared to be directly involved in both substrate and sulfa binding, based on homology to the Escherichia coli DHPS crystal structure. Thus, the results were consistent with the possibility that mutations in the P. carinii DHPS are responsible for some of the failures of sulfa/sulfone prophylaxis in AIDS patients.

Sequence Polymorphisms in the Pneumocystis carinii Cytochrome b Gene and Their Association with Atovaquone Prophylaxis Failure

Atovaquone (Mepron, 566c80) is an effective agent against Pneumocystis carinii, which probably acts by binding to cytochrome b and inhibiting electron transport. To assess the possibility that atovaquone resistance might be developing, the genes for the cytochrome b from P. carinii sp. f. carinii and P. carinii sp. f. hominis were partially sequenced. Eight of 10 patient isolates had cytochrome b genes with the same amino acid sequence. The P. carinii cytochrome b genes from 2 of 4 patients who had atovaquone prophylaxis failure contained mutations resulting in amino acid changes in one of the ubiquinone (coenzyme Q) binding sites (Qo). These mutations are homologous to mutations in other microorganisms that confer resistance to similar inhibitors. Variations in the sequence of the P. carinii cytochrome b gene suggest but do not prove the development of drug resistance.

Pneumocystis carinii cytochrome b mutations are associated with atovaquone exposure in patients with AIDS

This retrospective cohort study was conducted to determine whether Pneumocystis carinii cytochrome b gene mutations in patients with AIDS and P. carinii pneumonia (PCP) are associated with atovaquone exposure. Portions of the P. carinii cytochrome b genes that were obtained from 60 patients with AIDS and PCP from 6 medical centers between 1995 and 1999 were amplified and sequenced by using polymerase chain reaction. Fifteen patients with previous atovaquone prophylaxis or treatment exposure were matched with 45 patients with no atovaquone exposure. Cytochrome b coenzyme Q binding site mutations were observed in 33% of isolates from patients exposed to atovaquone, compared with 6% from those who were not (P=.018). There was no difference in survival 1 month after treatment between patients with or without cytochrome b mutations (P=.14). Thus, cytochrome b mutations are significantly more common in patients with AIDS and PCP with atovaquone exposure, but the clinical significance of these mutations remains unknown.

Interaction of rat Pneumocystis carinii and rat alveolar epithelial cells in vitro

During Pneumocystis carinii pneumonia, P. carinii trophic forms adhere tightly to type I alveolar epithelial cells (AECs). However, the manner in which the interaction between P. cariniiorganisms and AECs results in clinical pneumonia has not been explored. To investigate this interaction in vitro, we established a culture system using rat P. carinii and primary cultures of rat AECs. We hypothesized that binding of P. carinii to AECs would alter the metabolic, structural, and barrier functions of confluent AECs. Using fluorescently labeled P. carinii, we demonstrated that P. carinii bound to AECs in a dose-dependent manner. During P. carinii-AEC interaction, both the AECs and the P. carinii organisms remained metabolically active. Immunofluorescent staining demonstrated that AEC expression of the junctional proteins E-cadherin and occludin and the structural protein cytokeratin 8 were unaffected by P. carinii binding. To evaluate the effect of P. carinii on AEC barrier function, transepithelial resistance across AEC monolayers was measured during interaction with organisms. Culture with P. carinii did not result in loss of AEC barrier function but in fact increased AEC transepithelial resistance in a dose- and time-dependent manner. We conclude that the direct interaction of P. carinii with AECs does not disrupt AEC metabolic, structural, or barrier function. Therefore, we speculate that additional inflammatory cells and/or their signals are required to induce the epithelial derangements characteristic of P. carinii pneumonia.During Pneumocystis carinii pneumonia, P. carinii trophic forms adhere tightly to type I alveolar epithelial cells (AECs). However, the manner in which the interaction between P. carinii organisms and AECs results in clinical pneumonia has not been explored. To investigate this interaction in vitro, we established a culture system using rat P. carinii and primary cultures of rat AECs. We hypothesized that binding of P. carinii to AECs would alter the metabolic, structural, and barrier functions of confluent AECs. Using fluorescently labeled P. carinii, we demonstrated that P. carinii bound to AECs in a dose-dependent manner. During P. carinii-AEC interaction, both the AECs and the P. carinii organisms remained metabolically active. Immunofluorescent staining demonstrated that AEC expression of the junctional proteins E-cadherin and occludin and the structural protein cytokeratin 8 were unaffected by P. carinii binding. To evaluate the effect of P. carinii on AEC barrier function, transepithelial resistance across AEC monolayers was measured during interaction with organisms. Culture with P. carinii did not result in loss of AEC barrier function but in fact increased AEC transepithelial resistance in a dose- and time-dependent manner. We conclude that the direct interaction of P. carinii with AECs does not disrupt AEC metabolic, structural, or barrier function. Therefore, we speculate that additional inflammatory cells and/or their signals are required to induce the epithelial derangements characteristic of P. carinii pneumonia.

Connective tissue activation. XXI: Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP-III) growth factors

SummaryThe quantitative radiochemical methodology described in this report allows a major increase in information generation, increased experimental flexibility, improved statistical control, and increased diversity of information per culture. Other advantages relate to economies of technical time, supplies, cells, and test materials per individual culture.Microcultures of human synovial cells incorporate [14C]glucosamine into hyaluronic acid that accumulated primarily in the media and to a lesser extent in the cell mass. CTAP-I (from lymphoid cells), CTAP-III (from human platelets), PGE2, dibutyryl cAMP, and poly(I)·poly(C) markedly stimulated hyaluronate synthesis, whereas cortisol, cycloheximide, and tunicamycin inhibited stimulated synthesis. Time studies with cycloheximide indicated that translation, essential for the activation of synovial cells, was completed by 17 h postexposure to CTAP-I. Tunicamycin also seemed to inhibit CTAP-I induced activation primarily by interpering with translation; however, tunicamycin also caused modest post-translational inhibition of hyaluronate synthesis in activated adult human synovial cells.

Connective tissue activation. XXIII. Increased plasma levels of a platelet growth factor (CTAP-III) in patients with rheumatic diseases

Plasma levels of the CTAP-III antigen were measured by radioimmunoassay in 80 patients with rheumatic diseases. Patients with clear evidence of vasculitis usually exhibited increased plasma CTAP-III antigen. In both systemic lupus erythematosus and rheumatoid arthritis, there appeared to be a correlation between pCTAP-III values and other laboratory and clinical parameters of disease activity.

Distribution of DHPS mutations among ITS subtypes of P. carinii f. sp. hominis

 Corresponding author: Steven R. Meshnick – Telephone 734-647-2406; Fax: 734-764-3192; Email: meshnick@umich.edu J Eukaryot Microbiol, vol 48, Supplement, Jan/Feb 2002 issue. The IWOP7 Proceedings will still have a 2001 publication date. Sulfa drugs are widely used in the prophylaxis and treatment of Pneumocystis carinii pneumonia. Co-trimoxazole, the first-line agent, is a combination of sulfamethoxazole and trimethoprim but functions as sulfa monotherapy since P. carinii is apparently insensitive to trimethoprim. Dapsone, a sulfone, is a commonly used second-line agent. Both sulfamethoxazole and dapsone act by binding to dihydropteroate synthase (DHPS) (reviewed in references [2, 19]) (Fig. 1). Sulfa-resistant Pneumocystis carinii may be an emerging public health problem. However, since in vitro cultivation of human-derived organisms is not possible, molecular tools for determining resistance are needed. Genetic polymorphisms in the P. carinii DHPS were first reported in 1997 [11]. Two of the mutations, at positions 55 and 57, appear to be in the enzyme active site (Fig. 1, based on the E. coli structure [1]; MMDB id: 7741; PDB id: 1AJ0). These mutations have been associated with sulfa exposure, prophylaxis break-through, or treatment failure [4, 7, 9-11, 13-15, 18].

Evaluation of Sulfa Drugs against Recombinant Pneumocystis carinii Dihydropteroate Synthetase and In vivo

Sulfa drugs are potent and important anti-pneumocystis agents, but have a high incidence of adverse effects in AIDS patients. Although 15,000 sulfa drugs have been synthesized and dozens have been used in people, relatively few have been tested against P. carinii. In order to determine whether there are sulfa drugs which are better antipneumocystis agents than sulfamethoxazole and dapsone, we have been testing sulfa drugs in vitro, against recombinant P. carinii dihydropteroate synthetase, and in vivo. MATERIALS AND METHODS. Immunosuppression of latently infected rats, isolation of organisms, and enzyme and uptake assays were performed as previously described ( I ) . Rats were administered drug continuously via their drinking water for the full 6 weeks of immunosuppression. Exact doses were calculated by measuring daily water consumption. After 6 weeks, the rats were sacrificed, organisms were harvested, and the total number of cysts isolated from the lungs of each rat determined. Mice were treated either prophylactically or therapeutically as previously described (2). RESULTS AND DISCUSSION. Of the 44 sulfa drugs studied initially, 8 had ICSOs between 13 and 4 0 p M : sulfamethoxypyridazole, sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfathiourea, sulfadimethoxine, sulfisoxazole, and sulfaquinoxaline. In general, we found that sulfonamides (Fig. 1) were more potent than sulfones. The compounds with greatest potency tended to have heterocyclic substituents on the N1 position (R1). Substituents on the p-amino group (R2) ablated activity (I).