Reevaluation of the role of early trypsin activity in the transcriptional activation of the late trypsin gene in the mosquito Aedes aegypti.

In the female mosquito Aedes aegypti, trypsin expression is largely biphasic. Early trypsin synthesis, which is regulated at the translational level relative to feeding, peaks in the first few hours post-blood meal. Late trypsin expression is regulated at the transcriptional level, and peaks 18-24h post-blood meal. It was proposed that early trypsin activity released unknown factors during digestion of a meal that caused activation of transcription of the late trypsin gene. This connection between early trypsin activity and late trypsin expression was dependent on the fact that feeding a single trypsin inhibitor, soybean trypsin inhibitor (STI), which blocked early trypsin activity, also blocked late trypsin expression. We show in this study that feeding different trypsin inhibitors which effectively blocked early trypsin activity did not result in reduced late trypsin expression. We also found that a different lot of STI failed to cause inhibition of late trypsin transcription, although it was effective in inhibiting early trypsin activity. In addition, using RNAi methodology to reduce the level of early trypsin expression had no effect on the level of late trypsin expression. We conclude that early trypsin activity is not necessary for the transcriptional activation of late trypsin and that the previous results were due to the effect of a cytotoxic agent present in some, but not all preparations of STI.

The role of hemolymph proline as a nitrogen sink during blood meal digestion by the mosquito Aedes aegypti.

Mosquitoes utilize the amino acids derived from blood meal protein to produce egg proteins. But the amino acids can also be used to produce egg lipid or can be oxidized for energy production. These latter two processes result in the release of nitrogen as toxic ammonia. Therefore, amino acids must be processed in such a way that amino acid nitrogen can be incorporated into non-toxic waste products. Proline is the predominant amino acid in the hemolymph of the adult female mosquito Aedes aegypti. After feeding on albumin meal, hemolymph proline levels increased five-fold over unfed levels, reached maximal levels in the first hours after feeding and remained high through oviposition. Hemolymph proline levels increased as the concentration of protein in the meal increased. When starved of sugar for 24 h prior to feeding on an albumin meal, hemolymph proline levels increased four-fold over the proline levels of non-starved mosquitoes. Proline levels after feeding on a protein deficient in essential amino acids, pike parvalbumin, increased to twice the levels of albumin fed mosquitoes. Based on these observations, we propose that mosquitoes utilize proline as a temporary nitrogen sink to store ammonia arising from deamination of blood meal amino acid.

Fat metabolism in insects.

The study of fat metabolism in insects has received considerable attention over the years. Although by no means complete, there is a growing body of information about dietary lipid requirements, and the absolute requirement for sterol is of particular note. In this review we (a) summarize the state of understanding of the dietary requirements for the major lipids and (b) describe in detail the insect lipid transport system. Insects digest and absorb lipids similarly to vertebrates, but with some important differences. The hallmark of fat metabolism in insects centers on the lipid transport system. The major lipid transported is diacylglycerol, and it is carried by a high-density lipoprotein called lipophorin. Lipophorin is a reusable shuttle that picks up lipid from the gut and delivers it to tissues for storage or utilization without using the endocytic processes common to vertebrate cells. The mechanisms by which this occurs are not completely understood and offer fruitful areas for future research.

Lipid storage and mobilization in insects: current status and future directions.

In this paper we review the current status of research on fatty acid absorption and conversion to diacylglycerol in the midgut. We further discuss how diacylglycerol may leave the midgut and associate with lipophorin in hemolymph. We review the present understanding of the role of the lipid transfer particle and lipophorin receptors in lipid delivery between lipophorin and tissues. Finally, we discuss recent studies on the mobilization of diacylglycerol from the fat body in response to adipokinetic hormone. Several suggestions for exciting areas of future research are described.

A double blind, randomized, placebo-controlled phase II study to assess the safety and efficacy of orally administered SP-303 for the symptomatic treatment of diarrhea in patients with AIDS.

OBJECTIVE: The aim of this study was to investigate the safety and effectiveness of orally administered SP-303 in patients with AIDS and diarrhea. METHODS: This is a multicenter, phase II, randomized, double blind, placebo-controlled study. HIV-positive subjects with a history of a CD4 count <200 or an AIDS-defining illness were admitted to an inpatient study unit and screened for diarrhea defined as at least three abnormal (i.e., soft or watery) stools and >200 g of abnormal stool weight over a 24-h period. Subjects discontinued all antidiarrheal agents >24 h before enrollment. Stool samples were studied for routine pathogens. Subjects received 500 mg p.o. of SP-303 or placebo every 6 h for 96 h (4 days). Stool frequency and weights were recorded. Subjects were monitored for symptoms and side effects and were seen 1 wk later in follow-up. RESULTS: A total of 26 subjects received SP-303, and 25 received placebo. There were no significant demographic differences between treatment arms. A total of 41 subjects (80%) were receiving antiretroviral therapy and 39 subjects (77%) were receiving at least one protease inhibitor. Stool studies revealed no pathogens in 48 of 51 patients (94%). There were no serious adverse events or laboratory abnormalities. The SP-303 treatment group demonstrated a mean reduction from baseline stool weight of 451 g/24 h versus 150 g/24 h with placebo on day 4 of treatment (p = 0.14), and a mean reduction in abnormal stool frequency of three abnormal stools in 24 h versus two in 24 h in the placebo group (p = 0.30). Daily measures analysis over 4 days of treatment demonstrated that SP-303 subjects had a significant reduction in stool weight (p = 0.008) and abnormal stool frequency (p = 0.04) when compared to placebo-treated subjects. CONCLUSIONS: SP-303 is safe and well tolerated. These results suggest that SP-303 may be effective in reducing stool weight and frequency in patients with AIDS and diarrhea.

Lipophorin levels in the yellow fever mosquito, Aedes aegypti, and the effect of feeding.

High density lipophorin (HDLp) is the major lipid transport vehicle in insect hemolymph. Using an indirect ELISA, levels of HDLp were measured in the yellow fever mosquito, Aedes aegypti. The level of lipophorin, when normalized to the total weight of the insect, was similar in the different developmental stages. Starvation (access to water only) of adult females did not affect the level of HDLp nor its density when compared to sugar-fed females. On the other hand, blood feeding (of normally sugar-fed females) resulted in a three-fold increase of the HDLp level at 40 h after feeding. This increase was accompanied by a slight but significant increase in the density of HDLp at 24 h after feeding. Ingestion of a lipid-free protein meal or a lipid-supplemented protein meal induced changes in HDLp level and density that were comparable to those induced by ingestion of a blood meal. Ingestion of a blood meal, following starvation (access to water only) from the moment of adult emergence, did not induce an increase in HDLp level. The results presented indicate that, in contrast to other insect species, A. aegypti responds to an increased need for lipid transport in the hemolymph by increasing the amount of HDLp. Arch. Insect Biochem.

Lipid transfer from insect fat body to lipophorin: comparison between a mosquito triacylglycerol-rich lipophorin and a sphinx moth diacylglycerol-rich lipophorin.

Two insect lipoproteins, triacylglycerol-rich Aedes aegypti lipophorin and diacylglycerol-rich Manduca sexta lipophorin, were compared in their ability to load neutral lipid from fat body. When fat body of M. sexta was incubated in vitro with [3H]oleic acid, all radiolabeled fatty acids were esterified, predominantly to triacylglycerol. In A. aegypti fat body, however, half of the label remained as free fatty acids. When A. aegypti fat body was radiolabeled with [3H]glycerol, most of the radiolabel was incorporated in triacylglycerol. When either A. aegypti or M. sexta lipophorin was incubated with A. aegypti fat body, labeled with [3H]oleic acid, both lipophorins incorporated mainly radiolabeled free fatty acids, while almost no radiolabeled glycerides were transferred. When the same experiment was performed with A. aegypti fat body, radiolabeled with [3H]glycerol, very little transfer of radiolabeled glycerides was detected. In contrast, when either M. sexta or A. aegypti lipophorin was incubated with M. sexta fat body, both lipophorins incorporated neutral lipids, predominantly diacyglycerol. A. aegypti lipophorin incorporated half the amount of radiolabeled lipid, compared to M. sexta lipophorin. Lipophorins from both species were treated with triacylglycerol lipase of the yeast Candida cylindracea. Although this lipase readily delipidated M. sexta HDLp, it was not able to remove triacylglycerol from A. aegypti HDLp. The data presented suggest that, under the conditions used, lipid transfer from fat body to lipophorin in A. aegypti is not as efficient as in M. sexta.

Early trypsin, a female-specific midgut protease in Aedes aegypti: isolation, aminoterminal sequence determination, and cloning and sequencing of the gene.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. It plays an essential role in the transcriptional activation of the late trypsin form, the major midgut endoprotease involved in the blood meal digestion. Early trypsin is the most abundant midgut polypeptide isolated by benzamidine-sepharose affinity chromatography 3 h after feeding. The amino-terminal sequence of the early trypsin protein matches that of the 3a1 cDNA for a putative trypsinogen described by Kalhok et al. (Insect. Molec. Biol., 2, 71-79, 1993). The early trypsin cDNA was over expressed in Escherichia coli. Polyclonal antibodies generated against this recombinant protein were used to show that the enzyme was present in the midgut during the first 4 h after feeding. A 2.5 kb genomic clone of the early trypsin was isolated, mapped and subcloned. A 1.56 kb subclone, corresponding to 1303 bp of the upstream regulatory region and 265 bp of the coding region, was sequenced. The gene contains a 64 nucleotide intron which interrupts the codon for Val at position 18 of the protein. This Val is located toward the end of the putative signal sequence of the protein.

Aedes aegypti midgut early trypsin is post-transcriptionally regulated by blood feeding.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first hours after ingestion of a blood meal. Early trypsin gene expression was studied by Northern blot analysis. The early trypsin mRNA, absent in larvae, pupae and newly emerged females, reaches detectable levels at 24 h post-emergence and attains a maximum level at an adult age of 4-7 days. After the first week there is a decrease in the steady-state level of the transcript, but it remains readily detectable for up to a month after emergence. Despite the high levels of early trypsin mRNA present in the midgut of the unfed female, translation of the early trypsin mRNA occurs only after a blood or a protein meal. Early trypsin mRNA levels rapidly decrease during the first 24 h after feeding, but the steady-state level of the transcript rises again at the end of the blood digestion cycle (60 h), as the mosquito prepares for a second blood meal.

Clinical safety, tolerability, and pharmacokinetics of murine monoclonal antibody to human tumor necrosis factor-alpha.

The safety, tolerability, and pharmacokinetic profile of murine monoclonal antibody to human tumor necrosis factor-alpha (TNF alpha MAb) were evaluated in 20 uninfected patients at risk of sepsis and 16 septic patients. TNF alpha MAb was well tolerated in all patients, with no immediate or delayed signs of allergic reaction. During the 28-day evaluation, side effects included thrombocytosis (11), hepatic enzyme elevations (8), cardiac arrhythmias (3), and deaths (5). Each was attributed to the patient's severe underlying disease and not to TNF alpha MAb; however, a relationship between TNF alpha MAb and these events cannot be ruled out. The half-life was 52 h for a single infusion of TNF alpha MAb. Human antibody against TNF alpha MAb was observed in 13 (76.5%) of 17 phase IA patients and 10 of 10 phase IB patients and anti-idiotype antibodies in 11 (91.7%) of 12 phase IA patients and 2 (33.3%) of 6 phase IB patients. TNF alpha MAb should be evaluated as adjunctive therapy for patients with sepsis.

Therapy with antibody to tumor necrosis factor in sepsis.

Recent data indicate that proinflammatory cytokines mediate pathophysiological events during clinical sepsis. The cytokine tumor necrosis factor (TNF) has been closely associated with adverse outcome from sepsis, both in animal models and in the clinical setting. Accordingly, monoclonal antibodies with the capacity to neutralize TNF in vitro have been developed for evaluation as therapeutic agents in clinical sepsis. Preclinical studies in animal models of sepsis due to gram-negative as well as to gram-positive bacteria suggest that monoclonal antibodies to TNF may have potential as a therapeutic agent. Clinical trials to test this hypothesis are under way.

Tumor necrosis factor and the therapeutic potential of anti-tumor necrosis factor antibodies.

OBJECTIVE: To review the relationship of tumor necrosis factor (TNF) to clinical sepsis and the clinical potential of anti-TNF therapy in decreasing morbidity and mortality rates due to sepsis. DATA SOURCES: The international English language literature was reviewed, including animal studies and human clinical trials regarding TNF, anticytokine therapy, and sepsis. STUDY SELECTION: Studies which characterized the immunopharmacologic interactions between TNF and sepsis were emphasized. DATA EXTRACTION: This study specifically focused on experiments and clinical trials that directly involve the activity of TNF or anti-TNF antibodies, particularly but not limited to data derived from septic patients. DATA SYNTHESIS: The relationship between TNF and sepsis is described. Clinical aspects of anti-TNF therapy (timing, empiric use) are discussed. Phase I, II, and III trail of anti-TNF antibodies in clinical trials are reviewed. CONCLUSIONS: Current clinical strategies for sepsis therapy are only partially effective. Recent immunopharmacologic advancements have resulted in the identification of TNF as a pivotal proinflammatory cytokine mediator of sepsis. Animal studies demonstrate that anti-TNF therapy protects animals from the morbidity and mortality of sepsis. Phase I clinical studies of anti-TNF antibodies demonstrate the safety of monoclonal antibody therapy. The therapeutic application of anti-TNF antibodies in sepsis trials is ongoing.

Immunological perspectives in prevention and treatment of nosocomial pneumonia.

The high mortality associated with current therapeutic approaches to nosocomial pneumonia has motivated consideration of newer immunologic approaches to prevention or therapy of this infection. Serotype specific vaccines, hyperimmune immunoglobulins, and monoclonal antibodies have been developed for certain problematic pathogens. Pseudomonas aeruginosa has been the major focus of this approach, and trials of hyperimmune anti-Ps. aeruginosa globulins for treatment of pneumonia are underway. Broad-spectrum, anti-lipopolysaccharide antibody preparations have also been employed for prophylaxis of nosocomial pneumonia, but to date these trials have not been successful. Finally, anticytokine antibody therapy to reduce infection-initiated inflammatory lung damage is under consideration.

Safety, pharmacokinetics, and functional activity of human anti-Pseudomonas aeruginosa monoclonal antibodies in septic and nonseptic patients.

A mixture of five IgM human monoclonal antibodies (MAbs) against lipopolysaccharide antigens of Pseudomonas aeruginosa, plus a human IgG1 MAb against exotoxin A, were studied in 12 noninfected patients and 8 patients with P. aeruginosa bacteremia or pneumonia (or both). The preparation was well tolerated over a dose range of 0.3-1.2 ml/kg (0.75-3.0 mg/kg IgM protein). After a single infusion of 1.2 ml/kg (3.0 mg/kg IgM protein), serum antibody titers were boosted into therapeutic range, with serum half-lives ranging from 34 to 99 h. Also, opsonophagocytic activity in serum rose more than 1 log10 for all but one antibody. In no patient was an immunologic response against the MAb preparation detected.

Immunoglobulin therapy in infectious disease.

The role of immunoglobulin therapy in the prevention and treatment of infectious disease has been greatly expanded by the ability to administer immunoglobulins intravenously. Neonates, patients with AIDS, and bone marrow transplant recipients are beneficiaries of the advances being made in IgG therapy. Studies suggest that the synergistic effect of a combination of antibiotics and antibodies will be useful in the future. Other future directions for antibody therapy include development of monoclonal antibodies for treatment of sepsis. Also, clinical trials of monoclonal antibody against tumor necrosis factor are setting the stage for monoclonal antibody research directed increasingly to the anti-mediator concept.

Approach to therapy of respiratory infections in the critical care setting.

Therapy of pneumonia in the critical care setting includes intravenous antibiotics and supportive care. Since the etiologic agent of infection may not be clear, empiric broad-spectrum antibiotic regimens are often used. Combinations of beta-lactam and aminoglycoside agents are particularly popular regimens due to the high incidence of gram-negative bacillary and Staphylococcus aureus pneumonias in the critical care unit. Several new approaches to treatment of pneumonia in the critical care setting are being evaluated, including single-agent empiric coverage using a broad-spectrum beta-lactam agent; broad-spectrum quinolones, such as ciprofloxacin; intrabronchial aminoglycoside instillation therapy; and passive immune therapy with immunoglobulins and monoclonal antibodies.