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Pneumocystis is a fungal pathogen that can cause pneumonia in immunosuppressed hosts and subclinical infection in immunocompetent hosts. Mucosal-associated invariant T (MAIT) cells are unconventional lymphocytes with a semi-invariant T-cell receptor that are activated by riboflavin metabolites that are presented by the MHC-1b molecule MR1. Although Pneumocystis can presumably synthesize riboflavin metabolites based on whole-genome studies, the role of MAIT cells in controlling Pneumocystis infection is unknown. We used a co-housing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to characterize the response of MAIT cells to infection in C57BL/6 mice, and, using MR1−/− mice, which lack MAIT cells, to examine their role in clearing the infection. MAIT cells accumulated in the lungs of C57BL/6 mice during Pneumocystis infection and remained at increased levels for many weeks after clearance of infection. In MR1−/− mice, Pneumocystis infection was cleared with kinetics similar to C57BL/6 mice. Thus, MAIT cells are not necessary for control of Pneumocystis infection, but the prolonged retention of these cells in the lungs following clearance of infection may allow a more rapid future response to other pathogens.

Pneumocystis pneumonia continues to be a life-threatening infection in immunocompromised individuals. Dendritic cells are key antigen presenters to CD4+ T cells, which are critical to controlling Pneumocystis infection along with B cells. β-1,3 glucans are found in the cell wall of the cyst form of Pneumocystis. Glucans derived from other fungi have been shown to induce trained immunity. Therefore, we explored if Pneumocystis infection can induce a trained immunity response in C57Bl/6 (wild type) mice following exposure via a natural route of infection. We also tested activation-induced cytidine deaminase knockout mice (AID−/−), a deficiency that affects B cells, in the same infection model to determine whether a mutation known to affect the adaptive immune response can also alter the manifestation of trained immunity. We found that bone marrow derived dendritic cells (BMDCs) from C57BL/6 Pneumocystis exposed mice respond differently to homologous and heterologous antigen stimulation compared to BMDCs from naïve mice. Furthermore, although the differential response between cells from AID−/− exposed and naïve mice exists, specifically the TNFα response is opposite to that from C57BL/6 mice. In addition, when trained immunity was induced in BMDCs derived from naïve C57Bl/6 mice and AID−/− mice, the cytokine response was distinctive between the cells derived from C57BL/6 mice and AID−/− mice. These results suggest that not only can Pneumocystis infection induce trained immunity in BMDCs, this response is alterable by gene mutations traditionally thought to affect the adaptive immune response. Intramural Research Program of the NIH Clinical Center

Pneumocystis pneumonia (PCP) continues to be a life-threatening infection in HIV-infected patients, renal transplant patients, and other immunocompromised individuals. Dendritic cells are key antigen presenters to CD4+ T cells, which are critical to controlling Pneumocystis infection. β-1,3 glucans are found in the cell wall of the cyst form of Pneumocystis and are a known activator of innate immune cells via the dectin-1 receptor. β-glucans derived from other fungi have been shown to induce an enhanced “memory-like” immune response, termed trained immunity, in innate cells, including monocytes, macrophages, and dendritic cells. Therefore, we explored if Pneumocystis infection can induce an enhanced innate immune response in mice following exposure via a natural route of infection. We conducted mixing experiments using purified CD4+ T cells and dendritic cells from unexposed C57BL/6 mice and mice that spontaneously cleared infection. We found that CD4+ T cells from naïve mice exhibited a significantly greater proliferation after 5-day cultures when a crude Pneumocystis antigen was presented by CD11c+ dendritic cells from exposed mice compared to unexposed mice. In support of this, CD4+ T cells from C57BL/6 mice that were immunized with Freund’s adjuvant alone exhibited significantly greater proliferation when a crude Pneumocystis antigen was presented by either CD11c+ dendritic cells or CD11b+ macrophages from mice immunized with Pneumocystis antigen mixed with Freund’s adjuvant vs. adjuvant alone. These results suggest that Pneumocystis infection stimulates a memory-like innate immune response which may be important in controlling subsequent exposure to Pneumocystis, as well as exposure to other pulmonary pathogens.

The racemization of Z-Gly-Ser(Bzl)-OPcp was studied using the isotope effect to distinguish between enolization and 5(4H)-oxazolone mechanism. Labeled Z-Gly-L(α2H)-Ser(Bzl)-OPcp was racemized with NEt3 in THF to give kr2H = 570; similarly the unlabeled Z-Gly-L-Ser(Bzl)-OPcp yielded a racemization rate constant of krH = 720. The krH/kr2H ratio of 1.3 indicates that the serine dipeptide active ester derivatives racemize mainly through 5(4H)-oxazolone and to a lesser extent through the enolization mechanism, provided the 5(4H)-oxazolone racemizes much faster than it couples. 5(4H)-Oxazolone was prepared from Z-Gly-L-Ser(Bzl)-OH with DCC. Its first order racemization rate constant in THF is independent of its concentration, indicating that the racemization is not an intermolecular autoracemization. The unimolecular, that is intramolecular self-catalyzed, racemization is not possible based on molecular models. Therefore, it must be a solvent catalyzed bimolecular reaction. Its racemization was instantaneous with NEt3 while its coupling with H-Val-OMe was found to be 4 times 10-2M-1 s-1, that is kr± kc. The kc/kr values, which indicate the extent of racemization during coupling, are compared for benzyloxycarbonylamino and benzyloxycarbonylglycylamino acid active esters; the most vulnerable amino acids are His, Cys and Ser. The kc/kr values also indicate that for practical synthetic purposes the pentafluorophenyl esters should be preferred over other active esters to minimize racemization.

New 2,3,5,6-tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second-order kinetics showed similarly large kcoupling/kracemization ratios for both tetra- and pentafluorophenyl esters. In particular, the use of 2,3,5,6-tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N-tert.-butyloxycarbonyl-Nim-benzyl-histidine.

baseline viral load, history of opportunistic infection, co-morbidities, and age. Potential mechanisms underlying the low-level regeneration of CD4 cells in immune non-responders include deficiencies in the regeneration of central memory CD4 cells and excessive apoptosis8. Increased CD4 and CD8 T cell activation at baseline has also been correlated with poor immune responses to HAART9. In one early observational study, no association was found between a specific antiretroviral regimen and the presence of discordant responses5. In recent years, however, a number of reports have described CD4 declines or attenuated gains despite viral suppression in patients receiving a combination of didanosine and tenofovir10. While co-administration of these two agents has been shown to increase the ddI serum concentration, thereby increasing the risk of ddI-associated toxicities, the exact mechanisms leading to the CD4 effects are uncertain. The effect is diminished when a lower ddI dose is used11. Failure to increase CD4 counts may also be associated with additive myelotoxicity of antiretroviral drugs and therapies being administered for the prevention or treatment of opportunistic infection, as, for example, the additive myelosuppression of zidovudine (AZT) and trimethoprim/sulfamethoxazole (TMP/SMX). It has been postulated that ongoing, low-level viral replication, occurring below the limits of assay detection, may contribute to slowed CD4 T-cell recovery. Consequently, one approach has been to change or intensify antiretroviral regimens in an effort to more fully suppress viral replication and allow immune recovery, though available data do not support this practice. Alternative strategies aimed at improving the HIV-induced immunodeficiency utilize immune-based therapies. Potential approaches that have been studied over the years include therapy with cytokines such as interleukin-2 (IL-2), IL-12, IL-7, interpheron alpha, interpheron gamma and GM-CSF, as well as cell-transfer approaches using unmodified or genetically modified CD4 or CD8 cells. Immunosuppressive medications aiming to reduce high level immune activation thought to underlie CD4 cell turnover and death have also been studied, including cyclosporine A, anti-TNF monoclonal antibodies (etanercept), and mycophenolate mofetil. The best-studied and most promising immunotherapy agent for HIV infection that has been investigated to date is IL-2. IL-2 is a cytokine produced by T lymphocytes that is known to promote proliferation and modulate the secretory capacity of lymphocytes, including T, B, and natural killer cells. Numerous randomized trials have demonstrated that intermittent subcutaneous or intravenous administration of cycles of IL-2, in combination with antiHighly active antiretroviral therapy (HAART) has dramatically reduced the morbidity and mortality associated with HIV infection. In most patients receiving HAART, suppression of HIV replication is accompanied by recovery of CD4 T lymphocytes to near normal levels and a substantial reversal of HIV-associated immunologic defects, as is best reflected by the marked decrease in HIV-related opportunistic complications. However, immunologic recovery varies greatly between patients, and a significant proportion of those treated experience only small improvements in CD4 cell counts despite virologic suppression on HAART. These patients, who maintain virologic suppression on HAART but fail to have significant gains in CD4 T-cell counts, are referred to as immunologic non-responders. Estimates of the frequency of immunologic non-response vary depending on the definition used. Based on a number of cohort studies, 10% to 20% of treated patients fail to have an adequate CD4 count increase (e.g > 100 cells/mm3 increase over baseline or an increase to > 200-300 cells/mm3) after 6-12 months of effective HAART1-7. Whereas some patients with a poor initial immune response despite virologic suppression will go on to have an immunologic response over time, others will be stalled at a threshold that potentially puts them at increased risk for opportunistic complications. Observational cohort studies suggest that such patients do in fact have a higher rate of new AIDSdefining events or death than complete responders, though these rates are less than is seen in patients who are both immunologic and virologic non-responders2,6,7. The pathogenesis of discordant responses is poorly understood. The immunologic response to HAART is influenced by a number of viral, host, and treatment-related factors. Immune recovery depends, in part, on the extent and duration of viral load reduction, although the minimum viral suppression necessary for improved immune function and CD4 recovery is debated. Numerous other factors may influence CD4 T cell and immune recovery, including the degree of immunosuppression at HAART initiation (e.g., baseline CD4 count), duration of infection

The drug 566C80 is an investigational hydroxynaphthoquinone that is active against Pneumocystis carinii in vitro and in animal models. Initial studies in humans indicate that 566C80 is safe and has adequate bioavailability after oral administration.We conducted an open-label trial of 566C80 in 34 adults with the acquired immunodeficiency syndrome (AIDS) and untreated pneumocystis pneumonia. All the patients had a partial pressure of arterial oxygen of at least 60 mm Hg while breathing room air. They were enrolled sequentially in three cohorts taking 566C80 at different dosages, all administered orally: 750 mg three times daily for 5 days, then twice daily for 16 days; 750 mg three times daily for 21 days; and 750 mg four times daily for 21 days.All 34 patients survived, and 27 (79 percent) were successfully treated with 566C80 alone. The mean partial pressure of oxygen in 33 patients was 78 mm Hg at entry and 93 mm Hg after the course of 566C80 (P less than 0.001). In five patients (15 percent) the drug was discontinued because of lack of response. In four patients (12 percent), the drug was discontinued because of toxicity (fever and rash in two patients each). In two of these, treatment was considered to have succeeded because 566C80 was not discontinued because of toxicity until after day 14. Five of the successfully treated patients had rashes that resolved despite continued therapy. In nine patients, serum alanine aminotransferase levels rose above 100 U per liter. During the first three months after the completion of therapy, pneumocystis pneumonia recurred in 4 of the 27 successfully treated patients, and another 3 patients had recurrences between month 3 and month 6 of follow-up. The mean (+/- SEM) steady-state plasma levels of 566C80 were similar in the three cohorts: 16.3 +/- 2.10, 20.4 +/- 2.48, and 18.9 +/- 3.08 micrograms per milliliter in the patients taking the drug twice daily, three times daily, and four times daily, respectively.From these preliminary data, the investigational compound 566C80 appears to be a safe, effective, and well-tolerated therapy for P. carinii pneumonia of mild-to-moderate severity in patients with AIDS.

1. 1. The general reactions produced by large subcutaneous or oral doses of the more active choline compounds can be reproduced by iontophoresis, using the same drugs. In addition, there are certain local manifestations which appear at the site of application. 2. 2. On the basis of our experiments there is no consistent, marked change in the basal metabolic rate resulting from the use of the choline compounds in the dosages indicated. 3. 3. Although it is possible to produce a marked drop in blood pressure by the use of several of these compounds, this is a transient effect. We have not seen a single patient who has maintained a lowered blood pressure from the use of these drugs. 4. 4. Iontophoresis with mecholyl has proved to be a palliative method for the treatment of arthritis. 5. 5. In peripheral vascular disease, where organic occlusion has been the major factor, the choline compounds have not been of marked value. 6. 6. In vascular disease in which spasm is the major factor, the use of the choline compounds, as outlined, has proved helpful. 7. 7. A preliminary report on the successful treatment of long-standing chronic varicose ulcers in presented.

New 2,3,5,6-tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second-order kinetics showed similarly large kcoupling/kracemization ratios for both tetra- and pentafluorophenyl esters. In particular, the use of 2,3,5,6-tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N-tert.-butyloxycarbonyl-Nim-benzyl-histidine.

[Ser1], (32), [d-Ser1]- (29), [Ser1, Leu8]- (31), and [d-Ser1, Leu8] angiotensin II (30) were synthesized by a repetitive method in solution using new protected amino acid 2,3,5,6-tetrafluorophenyl active esters. 32 and 29 were agonists, and 31 and 30 were specific antagonists to angiotensin II (AII) receptors determined by the rabbit aortic strip (RAS) and rat blood pressure (RBP) assays. It was found that the hydroxymethyl side chains of serine and D-serine in position-l has an important influence on the agonistic activity of the analogues. The pressor activities of 32 and 29 were 129 and 314%, respectively, as potent as AII. On the other hand, 31 and 30 were effective in antagonizing the AII-induced contraction of RAS and rise in RBP.

A T cell clone (ACH-2) derived from T cells infected with HIV-1 was found to produce HIV-1 in response to stimulation with a monokine-enriched supernatant prepared by culturing human monocyte/macrophages with bacterial LPS (LPS-MO SN). Monokine induction of ACH-2 cells resulted in augmented virus production reflected by an increase in reverse transcriptase activity and in the synthesis of all major viral proteins. Examination of the cells by indirect immunofluorescence revealed that 10 to 15% of uninduced cells constitutively expressed HIV proteins, whereas 100% showed positive immunofluorescence in response to LPS-MO SN. This induction of virus by LPS-MO SN resulted in approximately a 100-fold increase of infectious virus production over uninduced ACH-2 cells. LPS alone could not induce HIV-1 expression, whereas LPS-MO SN resulted in the greatest virus expression. Cell separation studies confirmed the source of the inducing factor(s) to be cells bearing the mature monocyte/macrophage marker, Leu M3. Biochemical fractionation of the LPS-MO SN suggested that one or more factors, having apparent Mr of approximately 45 kDa, were involved in this induction. Absorption of the LPS-MO SN with immunoaffinity gels specific for human TNF-alpha was shown to completely remove the HIV inducing activity for the ACH-2 cell line.

To increase understanding of Pneumocystis carinii and its interaction with its hosts, Ag specific for rodent and human P. carinii were identified by the immunoblot method after PAGE of P. carinii organism extracts. The m.w. of the major Ag of rat P. carinii were 45,000, 110,000, and a broad band of 49,000 to 64,000, and of human P. carinii were 22,000, 24,000, and a broad band of 35,000 to 45,000 daltons. Human and rat pneumocystis were not antigenically identical. Specific antibodies against rat P. carinii Ag were found in 18 of 79 rats by the immunoblot method. Specific antibodies against human P. carinii Ag were found in 32 of 33 adult human sera, but in only 1 of 8 sera from infants and children. Specific antibodies were found in sera of 13 of the 14 adults with no history of P. carinii pneumonia, and all 19 patients with recently diagnosed P. carinii pneumonia, including 9 patients with P. carinii pneumonia associated with AIDS. The results of this study support previous suggestions that a large proportion of adults have been exposed to P. carinii and provide a basis for the further investigations of host-P. carinii interactions.