Leukocyte Membrane Enzymes Play the Cell Adhesion Game.

For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.

CD157 and Brain Immune System in (Patho)physiological Conditions: Focus on Brain Plasticity.

Ectoenzyme and receptor BST-1/CD157 has been considered as a key molecule involved in the regulation of functional activity of cells in various tissues and organs. It is commonly accepted that CD157 catalyzes NAD+ hydrolysis and acts as a component of integrin adhesion receptor complex. Such properties are important for the regulatory role of CD157 in neuronal and glial cells: in addition to recently discovered role in the regulation of emotions, motor functions, and social behavior, CD157 might serve as an important component of innate immune reactions in the central nervous system. Activation of innate immune system in the brain occurs in response to infectious agents as well as in brain injury and neurodegeneration. As an example, in microglial cells, association of CD157 with CD11b/CD18 complex drives reactive gliosis and neuroinflammation evident in brain ischemia, chronic neurodegeneration, and aging. There are various non-substrate ligands of CD157 belonging to the family of extracellular matrix proteins (fibronectin, collagen I, finbrinogen, and laminin) whose activity is required for controlling cell adhesion and migration. Therefore, CD157 could control structural and functional integrity of the blood-brain barrier and barriergenesis. On the other hand, contribution of CD157 to the regulation of brain development is rather possible since in the embryonic brain, CD157 expression is very high, whereas in the adult brain, CD157 is expressed on neural stem cells and, presumably, is involved in the neurogenesis. Besides, CD157 could mediate astrocytes' action on neural stem and progenitor cells within neurogenic niches. In this review we will summarize how CD157 may affect brain plasticity acting as a molecule at the crossroad of neurogenesis, cerebral angiogenesis, and immune regulation.

CD157: From immunoregulatory protein to potential therapeutic target.

CD157/BST1 glycosylphosphatidylinositol-anchored glycoprotein is an evolutionary conserved dual-function receptor and ß-NAD+-metabolizing ectoenzyme of the ADP-ribosyl cyclases gene family. Identified as bone marrow stromal cell and myeloid cell differentiation antigen, CD157 turned out to have a wider expression than originally assumed. The functional significance of human CD157 as an enzyme remains unclear, while it was well established in mouse models. Conversely, the receptor role of CD157 has been clearly delineated. In physiological conditions, CD157 is a key player in regulating leukocyte adhesion, migration and diapedesis. Underlying these functional roles is the ability of CD157 to bind with high affinity selected extracellular matrix components within their heparin-binding domains. CD157 binding to extracellular matrix promotes its interaction with ß1 and ß2-integrins and induces the organization of a multimolecular complex that is instrumental to the delivery of synergistic outside-in signals leading to optimal cell adhesion and migration, both in physiological and in pathological situations. CD157 also regulates cell adhesion and migration and is a marker of adverse prognosis in epithelial ovarian cancer and pleural mesothelioma. This review focuses on human CD157 expression and functions and provides an overview on its role in human pathology and its emerging potential as target for antibody-mediated immunotherapy.

Performance Characteristics of a Non-Fluorescent Aerolysin-Based Paroxysmal Nocturnal Hemoglobinuria (PNH) Assay for Simultaneous Evaluation of PNH Neutrophils and PNH Monocytes by Flow Cytometry, Following Published PNH Guidelines.

BACKGROUND: CD157 has been recently reported as a useful glycosylphosphatidylinositol (GPI)-linked marker for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with suspected paroxysmal nocturnal hemoglobinuria by flow cytometry as it targets both neutrophils and monocytes. The aim of this study is to test the feasibility of a non-fluorescent aerolysin (FLAER) approach and propose an alternative for laboratories, where FLAER is not available. METHODS: We validated a non-FLAER-based single-tube, 6-color assay targeting the GPI-linked structures CD157, CD24, and CD14. We determined its performance characteristics on 20 PNH patient samples containing a variety of clone sizes and compared results with a previously validated FLAER-based approach. RESULTS: Coefficient of variation (CV) for intra-/interassay precision analyses ranged from 0.1%/0.2% to 3.02%/7.58% for neutrophils and from 0.10%/0.3% to 5.39%/6.36% for monocytes. Coefficient of determination (r2 ) for linear regression analysis of PNH clones from 20 patients ranging from 0.06% to 99.7% was 0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from 0.06 to 0.2. CONCLUSION: Our results confirm very good performance characteristics for both intra- and interassay precision analyses, favorable correlation, and agreement between the FLAER and non-FLAER-based approaches, using the CD157 GPI marker. Our experience suggests that a rapid and cost-effective simultaneous evaluation of PNH neutrophils and monocytes by flow cytometry without using FLAER is possible in areas where FLAER may not be widely available. © 2016 International Clinical Cytometry Society.

CD157 at the intersection between leukocyte trafficking and epithelial ovarian cancer invasion.

CD157 is a member of the ADP-ribosyl cyclase gene family that is involved in the metabolism of NAD. CD157 behaves both as an ectoenzyme and as a receptor. Though CD157 is anchored to the membrane by a glycosylphosphatidylinositol moiety, which makes it unsuitable to transduce signals on its own, it exploits its localization in selected membrane microdomains and its proclivity to interact with integrins to accomplish receptor functions. Initially characterized as a stromal and myeloid antigen involved in the control of leukocyte adhesion, migration and diapedesis, CD157 was subsequently found to have a far wider distribution. In particular, CD157 was found to be expressed by epithelial ovarian cancer cells where it is involved in interactions among tumor cells, extracellular matrix proteins and mesothelium. The overall picture inferred from experimental and clinical observations is that CD157 is a critical player both in leukocyte trafficking and in ovarian cancer invasion and metastasis formation. In this review, we will discuss the biological mechanisms underpinning the role of CD157 in the control of leukocyte migration and ovarian cancer dissemination.

Ectoenzymes and innate immunity: the role of human CD157 in leukocyte trafficking.

CD157 is a glycosylphosphatidylinositol-anchored molecule encoded by a member of the CD38/ADP-ribosyl cyclase gene family, involved in the metabolism of NAD. Expressed mainly by cells of the myeloid lineage and by vascular endothelial cells, CD157 has a dual nature behaving both as an ectoenzyme and as a receptor. Although it lacks a cytoplasmic domain, and cannot transduce signals on its own, the molecule compensates for this structural limit by interacting with conventional receptors. Recent experimental evidence suggests that CD157 orchestrates critical functions of human neutrophils. Indeed, CD157-mediated signals promote cell polarization, regulate chemotaxis induced through the high affinity fMLP receptor and control transendothelial migration.

Molecular characterization of a novel cell surface ADP-ribosyl cyclase from the sea urchin.

The sea urchin is an extensively used model system for the study of calcium signalling by the messenger molecules NAADP and cyclic ADP-ribose. Both are synthesized by ADP-ribosyl cyclases but our molecular understanding of these enzymes in the sea urchin is limited. We have recently reported the cloning of an extended family of sea urchin ADP-ribosyl cyclases and shown that one of these enzymes (SpARC1) is active within the endoplasmic reticulum lumen. These studies suggest that production of messengers is compartmentalized. Here we characterize the properties of SpARC2. SpARC2 catalyzed both NAADP and cyclic ADP-ribose production. Unusually, the NAD surrogate, NGD was a poor substrate. In contrast to SpARC1, heterologously expressed SpARC2 localized to the plasma membrane via a glycosylphosphatidylinositol (GPI)-anchor. Transcripts for SpARC2 were readily detectable in sea urchin eggs and a majority of the endogenous membrane bound activity was found to be GPI-anchored. Our data reveal striking differences in the properties of sea urchin ADP-ribosyl cyclases and provide further evidence that messenger production may occur outside of the cytosol.

Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology.

The membrane proteins CD38 and CD157 belong to an evolutionarily conserved family of enzymes that play crucial roles in human physiology. Expressed in distinct patterns in most tissues, CD38 (and CD157) cleaves NAD(+) and NADP(+), generating cyclic ADP ribose (cADPR), NAADP, and ADPR. These reaction products are essential for the regulation of intracellular Ca(2+), the most ancient and universal cell signaling system. The entire family of enzymes controls complex processes, including egg fertilization, cell activation and proliferation, muscle contraction, hormone secretion, and immune responses. Over the course of evolution, the molecules have developed the ability to interact laterally and frontally with other surface proteins and have acquired receptor-like features. As detailed in this review, the loss of CD38 function is associated with impaired immune responses, metabolic disturbances, and behavioral modifications in mice. CD38 is a powerful disease marker for human leukemias and myelomas, is directly involved in the pathogenesis and outcome of human immunodeficiency virus infection and chronic lymphocytic leukemia, and controls insulin release and the development of diabetes. Here, the data concerning diseases are examined in view of potential clinical applications in diagnosis, prognosis, and therapy. The concluding remarks try to frame all of the currently available information within a unified working model that takes into account both the enzymatic and receptorial functions of the molecules.

CD157 is part of a supramolecular complex with CD11b/CD18 on the human neutrophil cell surface.

CD157 is a GPI-anchored cell surface glycoprotein expressed by human peripheral blood neutrophils. Cross-linking of CD157 induces intracellular Ca2+ mobilization and re-shaping in neutrophils, thus regulating their adhesive and migratory properties. Results obtained by immunolocalization and confocal microscopy indicate that CD157 lies in close proximity to the CD11b/CD18 complex which is strongly expressed on the activated neutrophil cell membrane where it plays a predominant role in adhesion. This study analyses the physical association between CD157 and CD18 in human neutrophils by co-immunoprecipitation experiments. The anti-CD157 monoclonal antibody RF3 co-precipitates CD18, and the anti-CD18 antibody TS1/18 co-precipitates CD157 from human neutrophil lysates. These results confirm that CD157 physically interacts with CD11b/CD18 complex in human neutrophils.

CD157 plays a pivotal role in neutrophil transendothelial migration.

Paracellular diapedesis, a key step in leukocyte recruitment to the site of inflammation, occurs at endothelial junctions and is regulated by highly coordinated interactions between leukocytes and endothelium. We found that CD157, a glycosylphosphatidylinositol-anchored ectoenzyme belonging to the NADase/ADP-ribosyl cyclase family, plays a crucial role for neutrophil diapedesis, because its ligation with specific monoclonal antibodies (both on neutrophils or endothelial cells) results in altered neutrophil movement on the apical surface of endothelium and, ultimately, in loss of diapedesis. Real-time microscopy revealed that CD157 behaves as a sort of compass during the interaction between neutrophils and endothelial cells; indeed, following CD157 ligation, neutrophils appear disoriented, meandering toward junctions where they eventually stop without transmigrating. These findings are relevant in vivo because CD157-deficient neutrophils obtained from patients with paroxysmal nocturnal hemoglobinuria are characterized by a severely impaired diapedesis.

CD38 induces apoptosis of a murine pro-B leukemic cell line by a tyrosine kinase-dependent but ADP-ribosyl cyclase- and NAD glycohydrolase-independent mechanism.

Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.

CD38 and CD157: biological observations to clinical therapeutic targets.

The application of molecular knowledge for developing new medical technologies is the goal of molecular medicine. Success in this area is highly dependent on the interaction of investigators from fields as diverse as biochemistry, cell biology, immunology, physiology, epidemiology, and physics, with an eye toward applying their insights and discoveries to improving human health. Such interdisciplinary approaches rarely find the common ground and language necessary to achieve this goal. Recently, a meeting of researchers studying the ectoenzymes CD38 and CD157 brought together insights into the regulation of calcium signaling, the metabolism of pyridine nucleotides by CD38 and CD157, and subsequent effects on immune function. Together, these discoveries were being applied to the development of novel therapeutics and diagnostics for myeloma and chronic lymphocytic leukemia. This issue of Molecular Medicine, featuring several short reviews based on a conference held in Turin, Italy, 10-12 June 2006, showcases the current state of this field and highlights some recent progress in molecular medicine.

CD38 and CD157 as receptors of the immune system: a bridge between innate and adaptive immunity.

This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage.

Requirement of 8-mercaptoguanosine as a costimulus for IL-4-dependent mu to gamma1 class switch recombination in CD38-activated B cells.

Mature B-2 cells expressing surface IgM and IgD proliferate upon stimulation by CD38, CD40 or lipopolysaccharide (LPS) and differentiate into IgG1-producing plasma cells in the presence of cytokines. The process of class switch recombination (CSR) from IgM to other isotypes is highly regulated by cytokines and activation-induced cytidine deaminase (AID). Blimp-1 and XBP-1 play an essential role in the terminal differentiation of switched B-2 cells to Ig-producing plasma cells. IL-5 induces AID and Blimp-1 expression in CD38- and CD40-activated B-2 cells, leading to mu to gamma1 CSR at DNA level and IgG1 production. IL-4, a well-known IgG1-inducing factor, does not induce mu to gamma1 CSR in CD38-activated B-2 cells or Blimp-1, while IL-4 induces mu to gamma1 CSR, XBP-1 expression, and IgG1 production expression in CD40-activated B-2 cells. Interestingly, the addition of 8-mercaptoguanosine (8-SGuo) with IL-4 to the culture of CD38-activated B cells can induce mu to gamma1 CSR, Blimp-1 expression, and IgG1 production. Intriguingly, 8-SGuo by itself induces AID expression in CD38-activated B cells. However, it does not induce mu to gamma1 CSR. These results imply that the mode of B-cell activation for extracellular stimulation affects the outcome of cytokine stimulation with respect to the efficiency and direction of CSR, and the requirements of the transcriptional regulator and the generation of antibody-secreting cells. Furthermore, our data suggest the requirement of additional molecules in addition to AID for CSR.

Production of calcium-mobilizing metabolites by a novel member of the ADP-ribosyl cyclase family expressed in Schistosoma mansoni.

ADP-ribosyl cyclases are structurally conserved enzymes that are best known for catalyzing the production of the calcium-mobilizing metabolite, cyclic adenosine diphosphate ribose (cADPR), from nicotinamide adenine dinucleotide (NAD(+)). However, these enzymes also produce adenosine diphosphate ribose (ADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP(+)), both of which have been shown to modulate calcium mobilization in vitro. We have now characterized a new member of the cyclase family from Schistosoma mansoni, a member of the Platyhelminthes phylum. We show that the novel NAD(P)(+) catabolizing enzyme (NACE) expressed by schistosomes is structurally most closely related to the cyclases cloned from Aplysia but also shows significant homology with the mammalian cyclases, CD38 and CD157. NACE expression is developmentally regulated in schistosomes, and the GPI-anchored protein is localized to the outer tegument of the adult schistosome. Importantly, NACE, like all members of the cyclase family, is a multifunctional enzyme and catalyzes NAD(+) glycohydrolase and base-exchange reactions to produce ADPR and NAADP(+). However, despite being competent to generate a cyclic product from NGD(+), a nonphysiologic surrogate substrate, NACE is so far the only enzyme in the cyclase family that is unable to produce significant amounts of cADPR (<0.02% of reaction products) using NAD(+) as the substrate. This suggests that the other calcium-mobilizing metabolites produced by NACE may be more important for calcium signaling in schistosomes. Alternatively, the function of NACE may be to catabolize extracellular NAD(+) to prevent its use by host enzymes that utilize this source of NAD(+) to facilitate immune responses.

Laser capture microdissection and single-cell RT-PCR without RNA purification.

Chronic infectious diseases of the central nervous system (CNS) are characterized by intrathecal synthesis of increased amounts of immunoglobulin G (IgG) directed against the agent that causes disease. In other inflammatory CNS diseases such as multiple sclerosis and CNS sarcoid, the targets of the humoral immune response are uncertain. To identify the IgGs expressed by individual CD38(+) plasma cells seen in human brain sections, we merged the techniques of laser capture microdissection (LCM) and single-cell RT-PCR. Frozen brain sections from a patient who died of subacute sclerosing panencephalitis (SSPE), were rapidly immunostained and examined by LCM to dissect individual CD38(+) cells. After cell lysis, we developed two techniques for reverse-transcription (RT) of unpurified total RNA in the cell lysates. The first method performed repeated and rapid freeze-thawing, followed by centrifugation of the cell lysate into tubes for subsequent RT. The second, more successful method performed RT in situ on detergent-solubilized cells directly on the cap surface; subsequent nested PCR identified heavy and light chain sequences expressed by two-thirds of individually isolated plasma cells. These techniques will streamline the identification of gene expression products in single cells from complex tissues and have the potential to identify IgGs expressed in the CNS of inflammatory diseases of unknown etiology.

[Morphological and functional characteristics of lymphokine-activated killer cells generated from the mononuclear cells of human peripheral blood].

The aim of this research was to study the morphological, functional and immunophenotypical characteristics of lymphokine-activated killer cells (LAKC) generated from the mononuclear cells (MNC) of healthy donors' peripheral blood at different time intervals after the cultivation with interleukin-2 (IL-2). LAKC had the appearance of large lymphoid cells of prolymphocyte and immunoblast type with highly pyroninophilic cytoplasm and electrone-microscopic features indicative of synthetic activity. LAKC were shown to intensely express activation antigens and adhesion molecules on their surface and to posess high cytotoxic potential in respect to tumor cells. Time-course of LAKC surface antigen expression corresponded to the changes of a proportion of activated cellular forms, generated from MNC of healthy donors' peripheral blood by incubation with IL-2. On the basis of these experimental findings, the usage of 3-5-day culture of LAKC could be recommended for the immunotherapy of malignant tumors.

Laser-capture microdissection of plasma cells from subacute sclerosing panencephalitis brain reveals intrathecal disease-relevant antibodies.

Increased IgG and oligoclonal bands are found in cerebrospinal fluid of humans with chronic infectious CNS disease. Studies have shown that these oligoclonal bands are antibodies directed against the agent that causes disease. Laser-capture microdissection was used to isolate individual CD38+ plasma cells from the brain of a patient with subacute sclerosing panencephalitis, and single-cell RT-PCR was used to analyze individual IgG heavy and light chains expressed by each cell. Based on overrepresented IgG sequences, we constructed functional recombinant antibodies (recombinant IgGs) and determined their specificities. Five of eight recombinant IgGs recognized measles virus, the cause of subacute sclerosing panencephalitis. These results demonstrate that overrepresented IgG sequences in postmortem brains can be used to produce functional recombinant antibodies that recognize their target antigens. This strategy can be used to identify disease-relevant antigens in CNS inflammatory diseases of unknown etiology.

Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice.

Here we report that a new nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse line harboring a complete null mutation of the common cytokine receptor gamma chain (NOD/SCID/interleukin 2 receptor [IL2r] gamma(null)) efficiently supports development of functional human hemato-lymphopoiesis. Purified human (h) CD34(+) or hCD34(+)hCD38(-) cord blood (CB) cells were transplanted into NOD/SCID/IL2rgamma(null) newborns via a facial vein. In all recipients injected with 10(5) hCD34(+) or 2 x 10(4) hCD34(+)hCD38(-) CB cells, human hematopoietic cells were reconstituted at approximately 70% of chimerisms. A high percentage of the human hematopoietic cell chimerism persisted for more than 24 weeks after transplantation, and hCD34(+) bone marrow grafts of primary recipients could reconstitute hematopoiesis in secondary NOD/SCID/IL2rgamma(null) recipients, suggesting that this system can support self-renewal of human hematopoietic stem cells. hCD34(+)hCD38(-) CB cells differentiated into mature blood cells, including myelomonocytes, dendritic cells, erythrocytes, platelets, and lymphocytes. Differentiation into each lineage occurred via developmental intermediates such as common lymphoid progenitors and common myeloid progenitors, recapitulating the steady-state human hematopoiesis. B cells underwent normal class switching, and produced antigen-specific immunoglobulins (Igs). T cells displayed the human leukocyte antigen (HLA)-dependent cytotoxic function. Furthermore, human IgA-secreting B cells were found in the intestinal mucosa, suggesting reconstitution of human mucosal immunity. Thus, the NOD/SCID/IL2rgamma(null) newborn system might be an important experimental model to study the human hemato-lymphoid system.

Early proliferation of CCR5(+) CD38(+++) antigen-specific CD4(+) Th1 effector cells during primary HIV-1 infection.

We investigated whether HIV-1 antigen-specific CD4(+) T cells expressed the viral coreceptor CCR5 during primary HIV-1 infection (PHI). In the peripheral blood of subjects with very early PHI (< 22 days after onset of symptoms), there was a 10- to 20-fold increase in the proportion of highly activated (CD38(+++)) and proliferating (Ki-67(+)) CD4(+) T cells that expressed CCR5(+), and were mostly T-cell intracellular antigen-1 (TIA-1)(+) perforin(+) granzyme B(+). Inthe same patient samples, CD4(+) T cells producing interferon (IFN)-gamma in response to HIV group-specific antigen (Gag) peptides were readily detected (median, 0.58%) by intracellular cytokine assay-these cells were again predominantly CD38(+++), Ki-67(+), and TIA-(++), as well as Bcl-2(low). On average, 20% of the Gag-specific CD4(+) T cells also expressed interleukin-2 (IL-2) and were CD127 (IL-7R)(+). Taken together, these results suggest that Gag-specific T-helper 1 (Th1) effector cells express CCR5 during the primary response and may include precursors of long-term self-renewing memory cells. However, in PHI subjects with later presentation, antigen-specific CD4(+) T cells could not be readily detected (median, 0.08%), coinciding with a 5-fold lower level of the CCR5(+)CD38(+++) CD4(+) T cells. These results suggest that the antiviral response to HIV-1 infection includes highly activated CCR5(+)CD4(+) cytotoxic effector cells, which are susceptible to both apoptosis and cytopathic infection with HIV-1, and rapidly decline.