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The reduction of disulfide bonds and their subsequent alkylation are commonplace in typical proteomics workflows. Here, we highlight a sulfhydryl-reactive alkylating reagent with a phosphonic acid group (iodoacetamido-LC-phosphonic acid, 6C-CysPAT) that facilitates the enrichment of cysteine-containing peptides for isobaric tag-based proteome abundance profiling. Specifically, we profile the proteome of the SH-SY5Y human cell line following 24 h treatments with two proteasome inhibitors (bortezomib and MG-132) in a tandem mass tag (TMT)pro9-plex experiment. We acquire three datasets─(1) Cys-peptide enriched, (2) the unbound complement, and (3) the non-depleted control─and compare the peptides and proteins quantified in each dataset, with emphasis on Cys-containing peptides. The data show that enrichment using 6C-Cys phosphonate adaptable tag (6C-CysPAT) can quantify over 38,000 Cys-containing peptides in 5 h with >90% specificity. In addition, our combined dataset provides the research community with a resource of over 9900 protein abundance profiles exhibiting the effects of two different proteasome inhibitors. Overall, the seamless incorporation of alkylation by 6C-CysPAT into a current TMT-based workflow permits the enrichment of a Cys-containing peptide subproteome. The acquisition of this “mini-Cys” dataset can be used to preview and assess the quality of a deep, fractionated dataset.

Thrombocytopenia is a common feature of antiphospholipid syndrome (APS) and rarely requires treatment. Here we present the case of a 71-year-old man hospitalized for severe immune thrombocytopenia (ITP) secondary to APS and concomitant SARS-CoV-2 infection. The patient was successfully treated with systemic corticosteroids, intravenous immunoglobulins, and plasma exchange (PEX). Few data are published on the use of plasma exchange in the treatment of thrombocytopenia in non-catastrophic APS. In the setting of acute infection when immunosuppressive therapies might be contraindicated, plasma exchange may be considered an effective therapeutic option. SARS-CoV-2 infection may be a trigger for a relapse of immune thrombocytopenia.

ObjectiveTo provide insights on emergent ethical tensions experienced by mental health practitioners during system re-organisation, which is sufficiently grounded in empirical data at the local level to inform policy on recovery at institutional and provincial levels.MethodEthnographic methods using narrative and critical phenomenological resources over 24 months.FindingsEveryday ethical tensions emerged at the confluence of different experiences of time, for example, how a context of increasing pressure to decrease patients’ length of stay at the hospital (service-defined time) challenged efforts to listen to and advocate for what mattered to patients (personal time) and maintain the integrity of interventions (clinical time). In this context, practitioners drew on clinical language and that of personal recovery to strategically ‘push back’, ‘play with’ or ‘take back’ time.DiscussionExamining everyday practices through ethnographic methods can illuminate the everyday ethical tensions that arise when mental health professionals and psychiatrists grapple with, often competing, goods. Critical phenomenological resources can help expand the structural considerations in empirical ethics, excavate underground practices and raise questions about the conceptual categories undergirding normative ethics. Experiencing-with practitioners in clinical contexts as they encounter and creatively resolve ethical tensions also propose a normative ethics of possibility, to help bridge the gap between empirical and normative ethics.ConclusionFocus on the relationship between policy, temporal practices and ethics suggests a reconfiguration of time and re-imagination of ethics in institutional settings in ways that can ultimately benefit patients and professionals alike.

Understanding and improving how diverse people work together is a core concern of applied social sciences. This article reports ethnographic observations on a participatory design project in which researchers and adults on the autism spectrum worked together on the design of a new technology—biomusic. Biomusic uses a smartphone application and a wearable sensor to measure physiological signals and translate them into auditory output. Ethnographers were involved in this project, both to facilitate eliciting perspectives of different stakeholders and to observe, record, and reflect on the process. This paper discusses the relationship between ethnography and participatory design in two ways. First, it describes the contribution of ethnography to achieving the goals of participatory design. Second, it draws on ethnographic observations to highlight different strategies people with and without autism used to work together, including strategies put forth by the researchers, strategies already in place in the community, and strategies emerging from the intersection of both. These strategies created a space that was more accessible to many different types of people. Documenting the way that this group worked together challenged several stereotypes about autism and highlighted the role of autistic collaborators as agents.

Sample multiplexing-based proteomic strategies rely on fractionation to improve proteome coverage. Tandem mass tag (TMT) experiments, for example, can currently accommodate up to 18 samples with proteins spanning several orders of magnitude, thus necessitating fractionation to achieve reasonable proteome coverage. Here, we present a simple yet effective peptide fractionation strategy that partitions a pooled TMT sample with a two-step elution using a strong anion-exchange (SAX) spin column prior to gradient-based basic pH reversed-phase (BPRP) fractionation. We highlight our strategy with a TMTpro18-plex experiment using nine diverse human cell lines in biological duplicate. We collected three data sets, one using only BPRP fractionation and two others of each SAX-partition followed by BPRP. The three data sets quantified a similar number of proteins and peptides, and the data highlight noticeable differences in the distribution of peptide charge and isoelectric point between the SAX partitions. The combined SAX partition data set contributed 10% more proteins and 20% more unique peptides that were not quantified by BPRP fractionation alone. In addition to this improved fractionation strategy, we provide an online resource of relative abundance profiles for over 11,000 proteins across the nine human cell lines, as well as two additional experiments using ovarian and pancreatic cancer cell lines.

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Large portions of the world's arable acreage experience water stress on a regular basis. Improving crop productivity in such drought‐prone environments is a critical breeding objective. The goal of this study was to detect quantitative trait loci (QTL) associated with alfalfa (Medicago sativaL.) forage productivity during drought stress. Two first‐generation backcross (BC1) mapping populations (n= 253) derived from a cross between M. sativasubsp. sativaand M. sativasubsp. falcatawere used to develop an updated tetraploid (2n= 4x= 32) genetic linkage map constructed from 600 single‐dose allele molecular markers. Map lengths associated with the two populations were 1293 and 1049 cM, with an average marker density of 3.8 and 3.9 cM, respectively. Half‐sib families derived from 206 BC1individuals were evaluated for forage yield in seeded plots in seven water‐stressed environments in New Mexico and Oklahoma, USA. Significant genotype effects were detected within each population and environment. Interval mapping analysis identified 10 and 15 QTL that, respectively, improved or reduced forage yield during drought. Average phenotypic effects of each QTL on biomass yield ranged from 3 to 6% and the direction of these effects were generally consistent over environments. Desirable alleles identified in these parents may be suitable for marker‐aided introgression into elite populations to incrementally improve their forage productivity in water‐limited environments.

Polo-like kinases are important regulators of multiple mitotic events; however, how Polo-like kinases are spatially and temporally regulated to perform their many tasks is not well understood. Here, we examined the subcellular localization of the budding yeast Polo-like kinase Cdc5 using a functional Cdc5-GFP protein expressed from the endogenous locus. In addition to the well-described localization of Cdc5 at the spindle pole bodies (SPBs) and the bud neck, we found that Cdc5-GFP accumulates in the nucleus in early mitosis but is released to the cytoplasm in late mitosis in a manner dependent on the Cdc14 phosphatase. This Cdc5 release from the nucleus is important for mitotic exit because artificial sequestration of Cdc5 in the nucleus by addition of a strong nuclear localization signal (NLS) resulted in mitotic exit defects. We identified a key cytoplasmic target of Cdc5 as Bfa1, an inhibitor of mitotic exit. Our study revealed a novel layer of Cdc5 regulation and suggests the existence of a possible coordination between Cdc5 and Cdc14 activity.

Cdc55, a regulatory B subunit of the protein phosphatase 2A (PP2A) complex, plays various functions during mitosis. Sequestration of Cdc55 from the nucleus by Zds1 and Zds2 is important for robust activation of mitotic Cdk1 and mitotic progression in budding yeast. However, Zds1-family proteins are found only in fungi but not in higher eukaryotes. In animal cells, highly conserved ENSA/ARPP-19 family proteins bind and inhibit PP2A–B55 activity for mitotic entry. In this study, we compared the relative contribution of Zds1/Zds2 and ENSA-family proteins Igo1/Igo2 on Cdc55 functions in budding yeast mitosis. We confirmed that Igo1/Igo2 can inhibit Cdc55 in early mitosis, but their contribution to Cdc55 regulation is relatively minor compared with the role of Zds1/Zds2. In contrast to Zds1, which primarily localized to the sites of cell polarity and in the cytoplasm, Igo1 is localized in the nucleus, suggesting that Igo1/Igo2 inhibit Cdc55 in a manner distinct from Zds1/Zds2.Our analysis confirmed an evolutionarily conserved function of ENSA-family proteins in inhibiting PP2A-Cdc55, and we propose that Zds1-dependent sequestration of PP2A-Cdc55 from the nucleus is uniquely evolved to facilitate closed mitosis in fungal species.

Throughout the past decade, a possible role of eosinophils in blood coagulation and thrombosis has been suggested. We conducted a Pubmed (MEDLINE) search of case and series referring to any kind of thrombotic events described in three conditions characterised by persistent blood eosinophilia, i.e. the hypereosinophilic syndrome (HES), the Churg Strauss syndrome (CSS), and parasitic infestations from 1966 to date. One hundred and ninety-two articles were found regarding thrombotic events in HES and CSS, and 209 cases of thrombosis were extracted. One hundred and seventy- seven articles dealing with parasitic diseases and thrombosis were found, but only 15 manuscripts reporting thrombosis of unknown origin in 22 patients were selected. In HES, arterial thromboses were more frequent than in CSS (p0.006), representing almost half of the cases (45), while venous and mixed artero-venous thrombosis were respectively 28 and 27. In contrast, in CSS there was a predominance of venous thrombosis (56, p0.006), with arterial thrombosis representing 38 of total thrombotic events, and mixed thrombosis being the least frequent (4). The higher incidence of arterial thrombosis in HES patients can be explained by the common cardiac involvement (64 of patients). In the 22 patients with parasitoses and thrombosis, 15 had arterial thrombosis (68) and 7 had venous thrombosis (32 ). Literature analysis shows that there are numerous reports of thrombotic events in patients with eosinophil-related disorders supporting a role for eosinophils in thrombosis. This observation raises the problem of prevention and treatment of thromboembolism particularly in HES and CSS patients.

Rosacea is a common skin disease with a high impact on quality of life. Characterized by erythema, edema, burning pain, immune infiltration, and facial skin fibrosis, rosacea has all the characteristics of neurogenic inflammation, a condition induced by sensory nerves via antidromically released neuromediators. To investigate the hypothesis of a central role of neural interactions in the pathophysiology, we analyzed molecular and morphological characteristics in the different subtypes of rosacea by immunohistochemistry, double immunofluorescence, morphometry, real-time PCR, and gene array analysis, and compared the findings with those for lupus erythematosus or healthy skin. Our results showed significantly dilated blood and lymphatic vessels. Signs of angiogenesis were only evident in phymatous rosacea. The number of mast cells and fibroblasts was increased in rosacea, already in subtypes in which fibrosis is not clinically apparent, indicating early activation. Sensory nerves were closely associated with blood vessels and mast cells, and were increased in erythematous rosacea. Gene array studies and qRT-PCR confirmed upregulation of genes involved in vasoregulation and neurogenic inflammation. Thus, dysregulation of mediators and receptors implicated in neurovascular and neuroimmune communication may be crucial at early stages of rosacea. Drugs that function on neurovascular and/or neuroimmune communication may be beneficial for the treatment of rosacea.

A novel, general approach to chemical inactivation of retroviruses was used to produce inactivated simian immunodeficiency virus (SIV) particles with functional envelope glycoproteins. Inactivated virions of three different virus isolates (SIVmne E11S, SIVmac239, and SIVmac239 g4,5), prepared by treatment with 2,2′-dithiodipyridine (aldrithol-2, AT-2), were not detectably infectious, in vitro or in vivo. Immunization of pigtailed macaques with inactivated SIVmne E11S particles, without adjuvant, induced both humoral and cellular immune responses. Four of six animals immunized with the inactivated particles did not show measurable SIV RNA in plasma (<100 copy Eq/ml) following intravenous challenge with pathogenic, homologous virus (SIVmne E11S), compared to peak values of ≥106 copy Eq/ml in challenged SIV-naive control animals (p = 0.0001). Despite the absence of measurable viral RNA in plasma in these animals, culturable virus and viral DNA were initially detectable in blood and lymph node specimens; in contrast to control animals, SIV DNA could no longer be detected in PBMC by 10 weeks postchallenge in five of six SIV-immunized animals (p = 0.0001). However, vaccines did not resist a sequential rechallenge with the heterologous pathogenic virus SIVsm E660. AT-2-inactivated virus with functional envelope glycoproteins is a novel class of vaccine immunogen and was noninfectious, under conditions of rigorous in vivo challenge, and induced both binding and neutralizing antibody responses, along with cellular immune responses. Results suggest that immunization facilitated effective containment of pathogenic homologous challenge virus. With further optimization, AT-2-inactivated viral particles may be a useful class of immunogen in the development of a vaccine to prevent AIDS.

ABSTRACTTransient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8+lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8+cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8+cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

ABSTRACTMolecular clones were constructed that express nucleocapsid (NC) deletion mutant simian immunodeficiency viruses (SIVs) that are replication defective but capable of completing virtually all of the steps of a single viral infection cycle. These steps include production of particles that are viral RNA deficient yet contain a full complement of processed viral proteins. The mutant particles are ultrastructurally indistinguishable from wild-type virus. Similar to a live attenuated vaccine, this approach should allow immunological presentation of a full range of viral epitopes, without the safety risks of replicating virus. A total of 11 Macaca nemestrinamacaques were inoculated with NC mutant SIV expressing DNA, intramuscularly (i.m.) in one study and i.m. and subcutaneously in another study. Six control animals received vector DNA lacking SIV sequences. Only modest and inconsistent humoral responses and no cellular immune responses were observed prior to challenge. Following intravenous challenge with 20 animal infectious doses of the pathogenic SIV(Mne) in a long-term study, all control animals became infected and three of four animals developed progressive SIV disease leading to death. All 11 NC mutant SIV DNA-immunized animals became infected following challenge but typically showed decreased initial peak plasma SIV RNA levels compared to those of control animals (P= 0.0007). In the long-term study, most of the immunized animals had low or undetectable postacute levels of plasma SIV RNA, and no CD4+T-cell depletion or clinical evidence of progressive disease, over more than 2 years of observation. Although a subset of immunized and control animals were boosted with SIV(Mne) proteins, no apparent protective benefit was observed. Immunization of macaques with DNA that codes for replication-defective but structurally complete virions appears to protect from or at least delay the onset of AIDS after infection with a pathogenic immunodeficiency virus. With further optimization, this may be a promising approach for vaccine development.

ABSTRACTTo better understand the viral and host factors involved in the establishment of persistent productive infection by primate lentiviruses, we varied the time of initiation and duration of postinoculation antiretroviral treatment with tenofovir {9-[2-(R)-(phosphonomethoxy)propyl]adenine}while performing intensive virologic and immunologic monitoring in rhesus macaques, inoculated intravenously with simian immunodeficiency virus SIVsmE660. Postinoculation treatment did not block the initial infection, but we identified treatment regimens that prevented the establishment of persistent productive infection, as judged by the absence of measurable plasma viremia following drug discontinuation. While immune responses were heterogeneous, animals in which treatment resulted in prevention of persistent productive infection showed a higher frequency and higher levels of SIV-specific lymphocyte proliferative responses during the treatment period compared to control animals, despite the absence of either detectable plasma viremia or seroconversion. Animals protected from the initial establishment of persistent productive infection were also relatively or completely protected from subsequent homologous rechallenge. Even postinoculation treatment regimens that did not prevent establishment of persistent infection resulted in downmodulation of the level of plasma viremia following treatment cessation, compared to the viremia seen in untreated control animals, animals treated with regimens known to be ineffective, or the cumulative experience with the natural history of plasma viremia following infection with SIVsmE660. The results suggest that the host may be able to effectively control SIV infection if the initial exposure occurs under favorable conditions of low viral burden and in the absence of ongoing high level cytopathic infection of responding cells. These findings may be particularly important in relation to prospects for control of primate lentiviruses in the settings of both prophylactic and therapeutic vaccination for prevention of AIDS.

Abstract It has been established that human skin grafted onto the nude mouse is able to regenerate after being subjected to a full-thickness wound. In the present work, we sought to determine the cells involved in the connective tissue repair process following superficial wounding. Two months after transplantation, superficial wounds were made at the center of the graft using mechanical dermabrasion. At various times thereafter, ranging from 2 days to 6 weeks, healing grafts were harvested and processed for immunohistological study with species-specific and cross-reacting antibodies directed against human or mouse antigens. The grafted human skin regenerated according to the following series of events. First, the human dermis underneath the scab became devoid of human fibroblasts while the surrounding human dermis preserved its own characteristics. The TUNEL reaction on early-phase healing wounds indicated that apoptosis occurred steadily within this area and could be the mechanism by which cells disappeared. Moreover, cell death was reduced when the wound was covered with an occlusive dressing. The human dermis beneath the wound was then invaded by mouse cells which deposited type I collagen on the human extracellular matrix and produced mouse granulation tissue at the surface above it. Human keratinocytes migrated over the mouse granulation tissue to reconstruct the epidermis. Eventually, the mouse granulation tissue was progressively invaded by human fibroblasts, which formed a human neodermis. The overall process appeared to depend upon several successive epithelial-mesenchymal interactions, which were not species-specific. This suggests that myofibroblasts arise from a specific subpopulation of fibroblasts, probably located at the interface between the dermis and adipose tissue, and that the granulation tissue is eventually remodeled by another population of fibroblasts present in the human dermis.:

Phorbol myristate acetate (PMA) treatment of an EL‐4 thymoma cell line (EL‐4farrar) induced secretion of a factor that inhibited intracellular killing of Leishmania majoramastigotes by activated macrophages. Analysis of the cytokines produced by EL‐4 cells after PMA stimulation identified interleukin‐2 (IL‐2, 2500 U/ml), IL‐4 (1280 U/ml), interferon‐γ(IFN‐γ; 100 U/ml), and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF; 50 U/ml). Neither tumor necrosis factor nor transforming growth factor β(TGF‐β) was detected. Each of the cytokines present in EL‐4 fluids was assessed for capacity to activate macrophages for destruction of parasites or to suppress intracellular killing. IFN‐γand GM‐CSF both activated macrophages to kill Leishmania; I L‐2 and IL‐4 had no activity for induction of this anti‐microbial effector function. IL‐2 and IL‐4 were tested for their capacity to inhibit lymphokine‐ or IFN‐γ‐induced destruction of L. majorby macrophages: IL‐4 was ineffective, but IL‐2 markedly suppressed the activation of macrophages for intracellular killing. Addition of ≥ 10 U/ml of IL‐2 at the time of infection, or up to 4 h before, blocked up to 100% of the capacity of activated macrophages to kill intracellular amastigotes. Immunoaffinity treatment of EL‐4 fluids with anti‐IL‐2 antibody resulted in >80% reduction in suppression of intracellular killing. The suppressive effects of IL‐2 were not direct, but mediated by TGF‐β. IL‐2 induced resident peritoneal macrophages to secrete >5000 pg/ml TGF‐β1, a quantity that is > 500‐fold higher than constitutive background levels (20–40 pg/ml) and is sufficient to block intracellular killing activities. This increase in secretion of TGF‐βwas not dependent increases in TGF‐β1 mRNA. Treatment of cultures with EL‐4 fluids or recombinant IL‐2 in the presence of antibody to TGF‐β1 blocked the suppressive activity of both. Thus, IL‐2 was the major suppressor factor in EL‐4 fluids, and it acted indirectly through the induction and autocrine action of TGF‐β. J. Leukoc. Biol.55: 81–90; 1994.

There is increasing evidence that cytokines play an important role in the pathogenesis of uveitis. The aim of this study was to determine the presence of cytokines in the aqueous humor (AH) of patients with chronic idiopathic uveitis (CU). Patients with uveitis (n=10) were compared to those undergoing cataract surgery (n=1) for non-inflammatory eye diseases. ELISA's for the detection of cytokines (IL-1, IL-2, IL-6, TNF-α) in aqueous humor were developed that allowed the measurement of multiple cytokines present in low concentrations. Interleukin-6 was found to be elevated in the aqueous humor of two patients (20%) with CU, but in none of the controls. Interleukin-1-α, Interleukin-2 and TNF-α were not detected in the AH of patients or controls. TGF-ß was detected in the aqueous of all patients and controls, using a bioassay.

ABSTRACTWhole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2'-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4+target cells and mediated virus-induced, CD4-dependent “fusion from without” comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.

Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1.

Patients with hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) were treated with recombinant interferon alpha A (rIFN- alpha A). The binding of iodinated recombinant interferon-alpha to baseline samples of peripheral blood mononuclear cells (PBMCs) from the leukemia patients was compared with clinical responsiveness to rIFN- alpha A. HCL patients (8/10) responded to rIFN-alpha A therapy, whereas none (0/10) of the CLL patients studied responded. The PBMCs from the eight responsive HCL patients bound approximately twice as much iodinated interferon as the PBMCs from nonresponsive CLL patients. This difference was due to more high-affinity receptors per cell with no difference in the affinity of the interferon-receptor interaction. However, because PBMCs from HCL patients were larger than PBMCs from CLL patients, the cell surface receptor density was similar. The leukemic cells from one of the two nonresponsive HCL patients bound iodinated interferon similarly to the cells from the responsive HCL patients, whereas the leukemic cells from the other nonresponsive HCL patient bound considerably less. The rapidity of response of the HCL patients did not correlate with the level of binding of iodinated interferon. Our results suggest that the absolute number of interferon receptors per cell may be only one of several important parameters in the response to rIFN-alpha A therapy, and that the responsiveness of a particular lymphoproliferative disease or a particular patient to rIFN- alpha A therapy cannot be predicted or explained solely by the degree of interaction between IFN and its cell surface receptor.

In an effort to determine the impact of halothane anesthesia on certain human cell-mediated immune functions, normal, purified human monocytes and lymphocytes were exposed to halothane in vitro at varying concentrations for up to 8 hours. Subsequently, these human effector cells were analyzed for their ability to function in several cell-mediated immunologic assays. Natural killer cell activity against K-562 was unaffected by halothane in most of the donors tested. Similarly, the ability of purified monocytes to inhibit MBL-2 tumor cell growth was unchanged. Halothane appeared to decrease the proliferative response of lymphocytes to phytohemagglutinin (PHA) in approximately 50% of the normal donors tested. In contrast, the ability of monocytes to lyse antibody-coated red cell targets (ADCC) was unaffected by even maximal exposure to halothane. Of interest was the finding that human monocytes exposed to as low as 2% halothane anesthesia for 4 hours displayed a dramatic down-regulation of hydrogen peroxide (H2O2) release. Since it is known that hydrogen peroxide and other incompletely reduced forms of oxygen secreted by monocytes can play a major role in the antimicrobial, antitumor, and inflammatory functions of these cells, this finding may help explain the enhanced susceptibility of post-operative patients to infections.

Patients with hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) were treated with recombinant interferon alpha A (rIFN-αA). The binding of iodinated recombinant interferon-a to baseline samples of peripheral blood mononuclear cells (PBMCs) from the leukemia patients was compared with clinical responsiveness to rIFN-αA. HCL patients (8/10) responded to rIFN-αA therapy, whereas none (0/10) of the CLL patients studied responded. The PBMCs from the eight responsive HCL patients bound approximately twice as much iodinated interferon as the PBMCs from nonresponsive CLL patients. This difference was due to more high-affinity receptors per cell with no difference in the affinity of the interferon-receptor interaction. However, because PBMCs from HCL patients were larger than PBMCs from CLL patients, the cell surface receptor density was similar. The leukemic cells from one of the two nonresponsive HCL patients bound iodinated interferon similarly to the cells from the responsive HCL patients, whereas the leukemic cells from the other nonresponsive HCL patient bound considerably less. The rapidity of response of the HCL patients did not correlate with the level of binding of iodinated interferon. Our results suggest that the absolute number of interferon receptors per cell may be only one of several important parameters in the response to rIFN-αA therapy, and that the responsiveness of a particular lymphoproliferative disease or a particular patient to rIFN-αA therapy cannot be predicted or explained solely by the degree of interaction between IFN and its cell surface receptor.

Bovine thymosin fraction 5, a partially purified thymic extract, has been employed in phase I and phase II clinical studies in immunodeficient cancer patients. Some improvement in cell-mediated immune functional parameters has been seen, notably an increase in percentages of peripheral blood T lymphocytes as detected by rosette formation with sheep erythrocytes. Thymosin-induced rosette-forming cell increases demonstrated in vitro prior to the institution of thymosin treatment generally were predictive of those patients who showed the in vivo rise in circulating T cells following thymosin administration. Although some patients exhibited clinical improvement, it is still too early to infer from these open studies whether or not these improvements can be attributed to thymosin treatment. Well-controlled randomized trials are in progress to objectively examine the clinical efficacy of thymosin fraction 5 therapy in cancer patients.

A review of the pleiotropic effects and numerous cell sources of interleukin 1 (IL 1) suggests it to be an important intercellular messenger. As such it is of considerable interest that normal human urine contains low molecular weight (4 kd and 2 kd) peptides with the thymocyte comitogenic activity of IL 1. These moieties may represent breakdown products of the usual 15 kd IL 1 peptide. Only the 2 kd but not the 4 kd urinary fraction exhibited the jibroblast proliferation activity of intact IL 1. Furthermore, anion exchange chromatography succeeded in separating the 2 kd fibroblast from the 2 kd thymocyte comitogenic activity. Consequently, determination of the portions of the IL 1 molecule that account for its pleiotropic biological effects will be greatly facilitated. Furthermore, the direct measurement of IL 1 activity in urine of patients with immunologic abnormalities, or with neoplastic or renal disease may prove valuable as a diagnostic and/or prognostic indicator.

Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze parallel reactions that are believed to be involved in the biosynthesis of lignin monomers. Antisera specific for alfalfa (Medicago sativaL.) COMT or CCOMT were raised against the enzymes expressed inEscherichia coli, and were used for immunolocalization studies in lignifying alfalfa stem tissue. Both COMT and CCOMT were localized to xylem parenchyma cells, as assessed by light microscopy and immunocytochemistry. Electron microscopy revealed that both enzymes were located in the cytoplasm of xylem parenchyma cells, and to a lesser extent, in the cytoplasm of phloem cells. There was no significant difference in the localization pattern of COMT and CCOMT, suggesting that the two enzymes may be part of a metabolic grid leading to production of lignin monomers in lignifying tissue of mature alfalfa stem internodes.

The amino acid sequence of a biologically active polypeptide isolated from calf thymus, termed thymosin alpha1, has been determined. Thymosin alpha1 is a heat stable, highly acidic molecule composed of 28 amino acid residues. This peptide is one of several present in thymosin fraction 5 that may participate in the regulation, differentiation and function of thymus-dependent lymphocytes (T cells). A nomenclature for the family of polypeptides present in thymosin fraction 5 is suggested.

Bovine thymosin fraction 5, a partially purified thymic extract, has been employed in phase I and phase II clinical studies in immunodeficient cancer patients. Some improvement in cell-mediated immune functional parameters has been seen, notably an increase in percentages of peripheral blood T lymphocytes as detected by rosette formation with sheep erythrocytes. Thymosin-induced rosette-forming cell increases demonstrated in vitro prior to the institution of thymosin treatment generally were predictive of those patients who showed the in vivo rise in circulating T cells following thymosin administration. Although some patients exhibited clinical improvement, it is still too early to infer from these open studies whether or not these improvements can be attributed to thymosin treatment. Well-controlled randomized trials are in progress to objectively examine the clinical efficacy of thymosin fraction 5 therapy in cancer patients. Thymosin fraction 5 contains several thymus-specific polypeptides which appear to act on lymphoid cells at a number of stages during their differentiation. Several of these individual polypeptides have been isolated, and one has been sequenced (thymosina1) and found to have immunoenhancing activity. Cancer patients may be viewed in many cases as exhibiting an “acquired” disease-related or treatment-related deficiency in cell-mediated immunity. This may render immunotherapeutic manipulations nonbeneficial and may in fact contribute to tumor progression and/or metastasis. By treating such patients with thymosin fraction 5 or the specific polypeptide(s) within fraction 5 required to allow the appearance of immunocompetent T lymphocytes, these patients may become amenable to immunotherapy, more resistant to infection with opportunistic organisms, and perhaps may develop endogenous antitumor defenses. La fraction 5 de la thymosine bovine, un extrait thymique partiellement purifié, a été utilisée dans des études cliniques (phases I et II) chez des cancéreux présentant des déficits immunitaires. Nous avons observé des améliorations de certains caractères fonctionnels d'immunité cellulaire, entre autres des augmentations de pourcentages de lymphocytes T dans le sang périphérique (mesuré par la formation de rosettes en présence de globules rouges de mouton). En général, l'observation in vitro, sous l'effet de la thymosine, d'une augmentation du nombre de cellules génératrices de rosettes, permet de prédire l'effet thérapeutique in vivo: les mÊmes malades voient, après administration de thymosine, s'élever leur nombre de cellules T circulantes. Bien que nous ayons constaté certaines améliorations cliniques, il est trop tÔt pour affirmer, sur la base de ces quelques essais, que c'est bien la thymosine qui a amélioré l'état des malades. Des études randomisées et contrÔlées sont en cours pour préciser ces premières appréciations. La fraction 5 de la thymosine contient des polypeptides spécifiques thymiques qui paraissent agir sur certains stades de la différenciation des lymphocytes. Plusieurs de ces polypeptides ont été isolés dans la fraction 5. L'un d'entre eux (thymosinea1) a une activité immunostimulante in vitro. On a pu déterminer la séquence d'acides aminés qui le composent. On peut admettre que de nombreux malades atteints de cancer présentent un déficit de l'immunité cellulaire, dépendant de la maladie ou du traitement. A cause de ce désordre, certaines thérapeutiques à visée immunologique peuvent Être délétères: elles peuvent favoriser la croissance et/ou la dissémination de la tumeur. En traitant ces patients avec la fraction 5 ou avec son (ou ses) polypeptide(s) spécifiques qui stimulent la formation de lymphocytes T immunocompétents, on peut espérer qu'ils deviendront justiciables d'une immunothérapie, qu'ils seront plus résistants aux infections occasionnelles et qu'ils développeront mÊme peut-Être des mécanismes endogènes de défense anticancéreuse.

An aqueous two-phase polymer system was used to isolate plasma membranes from a palpable mouse fibrosarcoma. The excised tumor tissue was washed with sterile saline and pushed through nylon screens of decreasing mesh size. This cell suspension was placed in Tris-buffered, isotonic sucrose plus MgSo4 and homogenized by nitrogen cavitation. A pellet was collected from the homogenate by low-speed centrifugation and was added to the aqueous two-phase polymer system. After several brief, low-speed centrifugations, the interfacial material between the polymer phases was collected. Data from enzyme and biochemical assays demonstrated that this fraction was plasma membrane. This method provided a high yield of the surface membrane in less than three hours.

An improved migration inhibitory factor (MIF) assay procedure is described which was developed for the study of tumor patient immunocompetence testing both on the clinical level and as an adjunct to basic studies of tumor immunology and the transfer of immunological capacity through immuno-genic RNA. A detailed description of a new migration chamber in which as many as eight capillaries can be simultaneously subjected to identical environmental conditions is presented, This feature reduces the variation among replicate tests to the point where significance can be ascribed to migration differences of ten percent or less. This chamber, along with the use of a cultured human lymphoblastoid tumor cell as the indicator cell, results in a very flexible, convenient, and sensitive direct or indirect assay for the detection of human migration inhibitory factor.

ABSTRACTWe tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4+T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4+T-cell depletion, consistent with mechanisms requiring productive infection.

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4(+) T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4(+) T-cell depletion, consistent with mechanisms requiring productive infection.

The article profiles the Southwest Michigan Black Heritage Society (SMBHS). According to the article, the SMBHS was established in October 2003 by residents of Kalamazoo, Michigan, who were concerned that African American history in southwest Michigan was not adequately recognized. It discusses several activities of the SMBHS including community workshops, oral history projects, and a book fair.

Lymphokine-activated killer (LAK) cells and interleukln-2 (IL-2) were administered by the ip route to patients with intra-abdominal malignancies. Pharmacokinetic studies of IL-2 revealed 10-to 100-fold higher concentrations of IL-2 in peritoneal fluid versus serum. Ip levels of IL-2 were maintained well above those required to generate and maintain LAK cells in vitro. LAK cell activity was detectable in the peritoneal fluid for the duration of each treatment cycle and did not disappear until IL-2 was discontinued. Detection of interferon-gamma (IFN-γ) in the peritoneal fluid of all patients was consistent with production in situ by activated lymphocytes. In some patients, low but detectable levels of IFN-γ were also found in the serum. In vivo activation of monocytes in the peritoneal fluid as measured by in vitro production of hydrogen peroxide was documented in the majority of patients. Neither interleukin-1 nor tumor necrosis factor-alpha was detected in the peritoneal fluid. We found no correlation between the presence or levels of IL-2, IFN-γ, or LAK cell lytic activity in peritoneal fluid or serum and response or nonresponse to therapy. [J Natl Cancer Inst 81:602–611, 1989]