Frontiers of robotic endoscopic capsules: a review.

Digestive diseases are a major burden for society and healthcare systems, and with an aging population, the importance of their effective management will become critical. Healthcare systems worldwide already struggle to insure quality and affordability of healthcare delivery and this will be a significant challenge in the midterm future. Wireless capsule endoscopy (WCE), introduced in 2000 by Given Imaging Ltd., is an example of disruptive technology and represents an attractive alternative to traditional diagnostic techniques. WCE overcomes conventional endoscopy enabling inspection of the digestive system without discomfort or the need for sedation. Thus, it has the advantage of encouraging patients to undergo gastrointestinal (GI) tract examinations and of facilitating mass screening programmes. With the integration of further capabilities based on microrobotics, e.g. active locomotion and embedded therapeutic modules, WCE could become the key-technology for GI diagnosis and treatment. This review presents a research update on WCE and describes the state-of-the-art of current endoscopic devices with a focus on research-oriented robotic capsule endoscopes enabled by microsystem technologies. The article also presents a visionary perspective on WCE potential for screening, diagnostic and therapeutic endoscopic procedures.

Antiviral profile of HIV inhibitors in macrophages: implications for therapy.

Macrophages (M/M) are identified as the second cellular target of HIV and a crucial virus reservoir. M/M are persistently infected cells and not susceptible to the HIV cytophatic effects typical of infected CD4+ T-lymphocytes. HIV replication in M/M is a crucial pathogenetic event during the whole course of the disease. Moreover, the dynamics of HIV-1 replication and cumulative virus production is quite different in M/M and CD4+ T-lymphocytes in the presence or in the absence of antiviral drugs. Thus, for their unique cellular characteristics, the activity of anti-HIV compounds could be different in M/M than in CD4+ T-lymphocytes. Indeed, nucleoside analogues inhibitors of HIV-reverse transcriptase (NRTIs) show potent antiviral activity in macrophages, although the limited penetration of these compounds in sequestered body compartments and the scarce phosphorylation ability of macrophages, suggest that a phosphate group linked to NRTIs may confer a greater anti-HIV activity in such cells. The antiviral activity of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in macrophages is similar to that found in CD4-lymphocytes. Interestingly, protease inhibitors (PIs), acting at post-integrational stages of virus replication, are the only drugs able to interfere with virus production and release from macrophages with established and persistent HIV infection. For these reasons, a careful analysis of the distribution of antiviral drugs, and the assessment of their activity in cells of macrophage lineage, represent key factors in the development of therapeutic strategies aimed to the treatment of the HIV-infected patients.

Macrophages: a crucial reservoir for human immunodeficiency virus in the body.

The replication of Human Immunodeficiency Virus (HIV) in cells of macrophage lineage represents a key pathogenetic event of the neurological damages typically found during the course of this disease. Macrophages are persistently infected cells and thus not susceptible to the cytophatic effect typical of infected activated CD4-lymphocytes. The resistance of macrophages to HIV infection is at least in part mediated by the autocrine production of the nerve growth factor (NGF), a neurokine able to sustain the survival of some cells of bone marrow origin, including monocyte-derived macrophages. This anti-apoptotic effect of NGF in HIV-infected macrophages can be even more relevant at the central nervous system level, where many cells are able to physiologically produce NGF, thus further increasing the survival of macrophages infected by HIV, and enhancing the damages that these cells may induce upon bystander neurons. The proapoptotic effect of soluble factors released by HIV-infected macrophages may heavily affect the survival and functions also of astrocytes, that in turn become unable to sustain neuronal homeostasis. Taken together, this information supports the importance of therapeutic attempts aimed at attacking virus replication in infected macrophages and/or to selectively eliminate these chronically infected and persistently virus-producing cells.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes.

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces < 10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death. Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.

Viral dynamics and mutations in the pol gene during primary HIV-1 infection.

The understanding of viral dynamics and appearance of mutations during primary infection could be useful for the design of an efficient therapy. For this reason a cohort of samples from naive primary patients was examined. The results pointed out that only a few secondary mutations in protease gene (having no effect on resistance) were found, while a single mutation conferring resistance to non-nucleosides inhibitors of reverse transcriptase was found both in plasma and cerebrospinal fluid of a patient. As both the protease secondary mutations and the single non nucleoside reverse transcriptase mutation map far from the catalytical sites of the enzymes, neither one is able to impair viral fitness. Overall data suggest that treated donors carrying resistant strains may be in part unable to transfer them to the recipient, and/or virus in the recipient tends to revert to wild type. These results should be taken into account in the planning of early HAART treatment of HIV infection.

Activities of masked 2',3'-dideoxynucleoside monophosphate derivatives against human immunodeficiency virus in resting macrophages.

The anti-human immunodeficiency virus (HIV) activity of aryloxyphosphoramidate protides of a number of anti-HIV nucleoside analogues was assessed in resting primary monocyte-macrophages (M/M). While 2',3'-dideoxythymidine (d4T), 2',3'-dideoxyadenosine (ddA), and 2',3'-dideoxy-2',3'-didehydroadenosine (d4A) protides showed an anti-HIV activity that was 25- to 625-fold greater than the parent nucleotides d4T, ddA, and d4A, respectively, other aryloxyphosphoramidate protides showed similar or even lower anti-HIV activities than their parent compounds. This variable anti-HIV effect is most likely related to the different dynamics of intracellular nucleoside monophosphate release from the protides. Our results indicate the potential advantage of therapeutic use of this approach for some nucleotide analogues to affect HIV replication in M/M, one of the major reservoirs of HIV in vivo.

Nerve growth factor is an autocrine factor essential for the survival of macrophages infected with HIV.

Nerve growth factor (NGF) is a neurotrophin with the ability to exert specific effects on cells of the immune system. Human monocytes/macrophages (M/M) infected in vitro with HIV type 1 (HIV-1) are able to produce substantial levels of NGF that are associated with enhanced expression of the high-affinity NGF receptor (p140 trkA) on the M/M surface. Treatment of HIV-infected human M/M with anti-NGF Ab blocking the biological activity of NGF leads to a marked decrease of the expression of p140 trkA high-affinity receptor, a concomitant increased expression of p75(NTR) low-affinity receptor for NGF, and the occurrence of apoptotic death of M/M. Taken together, these findings suggest a role for NGF as an autocrine survival factor that rescues human M/M from the cytopathic effect caused by HIV infection.

Relative potency of protease inhibitors in monocytes/macrophages acutely and chronically infected with human immunodeficiency virus.

The activity of three human immunodeficiency virus (HIV) protease inhibitors was investigated in human primary monocytes/macrophages (M/M) chronically infected by HIV-1. Saquinavir, KNI-272, and ritonavir inhibited the replication of HIV-1 in vitro, with EC50s of approximately 0.5-3.3 microM. However, only partial inhibition was achievable, even at the highest concentrations tested. Also, the activity of these drugs in chronically infected M/M was approximately 7- to 26-fold lower than in acutely infected M/M and approximately 2- to 10-fold lower than in chronically infected H9 lymphocytes. When protease inhibitors were removed from cultures of chronically infected M/M, production of virus rapidly returned to the levels found in untreated M/M. Therefore, relatively high concentrations of protease inhibitors are required to suppress HIV-1 production in chronically infected macrophages, and such cells may be a vulnerable point for the escape of virus in patients taking these drugs.

Clinical implications of HIV dynamics and drug resistance in macrophages.

Macrophages are widely recognized as the second major target of HIV in the body. The cellular characteristics of such resting cells markedly affect the dynamics of virus lifecycle, that is slower but far more prolonged that in lymphocytes. In addition, the limited concentrations of endogenous nucleotide pools in macrophages downregulate the enzymatic activity of reverse transcriptase. As a consequence, both the anti-HIV activity and the development of resistance to antiviral drugs in macrophages are substantially different than those found in activated lymphocytes. These peculiar characteristics of virus replication and efficacy of antiviral drugs in macrophages have a natural in vivo counterpart in extralymphoid tissues, where macrophages account for the majority of cells infected by HIV. Furthermore, the replication of HIV in macrophages of testis and central nervous system is far less affected by antiviral drugs than in lymph nodes, because of the presence of natural barriers that markedly diminish the concentration of such drugs. For all these reasons, HIV infection of macrophages should be taken into account in therapeutic strategies aimed to achieve an optimal therapeutic effect in all tissue compartments where the virus hides and replicates.

[Correlation between HIV-inhibiting drug activity in human macrophages and clinical outcome]. / Attività di farmaci inibitori del virus dell'immunodeficienza umana in macrofagi umani e correlazione con l'efficacia clinica.

PURPOSE: To assess the comparative efficacy of drugs inhibitors of human immunodeficiency virus (HIV) in human macrophages and lymphocytes, and to correlate the results with the clinical outcome. MATERIALS AND METHODS: Human primary macrophages and lymphocytes were infected with HIV in the presence of the following HIV inhibitors, all currently in clinical use: zidovudine, stavudine, zalcitabine, didanosine, lamivudine, PMEA, PMPA (all inhibitors of HIV reverse transcriptase), saquinavir and U-75875 (inhibitors of HIV protease). RESULTS: All reverse transcriptase inhibitors tested showed a markedly higher antiviral activity in macrophages than in lymphocytes. Also protease inhibitors have a substantial anti-HIV activity in macrophages, yet their efficacy is markedly diminished if the drugs are added to macrophage culture after HIV, that is when the virus has established a chronical infection. Under these experimental conditions, however, only protease inhibitors among all HIV-inhibitors in clinical use are able to decrease virus replication in chronically-infected macrophages. CONCLUSIONS: The results have strong clinical implications, due to the important role of macrophages in the pathogenesis of HIV infection. Macrophages are the major source of HIV at extralymphoid tissue levels, particularly in the central nervous system, where the blood-brain barrier strongly limits the penetration of antiviral drugs. For these reasons, only drugs, like stavudine and zidovudine, provided with good anti-HIV activity in macrophages, and reasonable barrier penetration have substantial chances to be effective in the central nervous system, and thus affect virus replication in a sanctuary where HIV hides and replicates out of the control of the immune system.

Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes.

Several anti-HIV drugs acting on different steps of virus replication were tested in our experimental model of primary monocyte/macrophages; the results were compared with the activity found in lymphocytes. Nucleoside analogues (AZT, ddI, ddC, d4T, PMEA, 3TC etc.) show greater activity in macrophages (M/M) than in lymphocytes. In particular, the EC50 of AZT, ddC, and ddI in M/M is 2- to 100-fold lower than that found in lymphocytes. This greater efficacy of nucleoside analogues in M/M depends on the enhancement of their chain-terminating activity by the low levels of endogenous deoxynucleoside-triphosphates (dNTP) usually found in resting cells such as M/M. Non-nucleoside reverse transcriptase inhibitors (NNRTI) do not act as chain terminators (thus their antiviral effect is not related to the intracellular concentrations of dNTP); as a consequence the activity of TSAO, HEPT, TIBO, and other NNRTI tested in M/M is similar to that found in lymphocytes. Regarding inhibitors of binding and fusion of HIV, we found that their anti-HIV activity is markedly decreased (or even nullified) when M/M are treated with cytokine activators of M/M function and enhancers of HIV replication. More relevant from a clinical standpoint, protease inhibitors are able to inhibit HIV replication in chronically infected macrophages (i.e., cells carrying the proviral genome already integrated in the host genome). All other inhibitors of late stage of virus life cycle tested (antisense-rev, anti-tat, interferon-alpha and -gamma, phosphorothioate analogues, GLQ-223, etc.) were totally inactive in chronically infected macrophages. The different effects of various classes of HIV inhibitors in lymphocytes and macrophages suggests that AIDS therapy should consider all aspects of the pathogenesis of HIV infection and must be restricted to drugs, or combinations of drugs, active against both lymphocytes and M/M in all body compartments where the virus hides and replicates.

Red blood cells mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to primary macrophages: efficiency metabolism and activity against human immunodeficiency virus or herpes simplex virus.

Red blood cells (RBC) may act as selective carriers of drugs to macrophages, an important reservoir of viruses such as human immunodeficiency virus (HIV) and herpes simplex virus type 1 (HSV-1). We therefore assessed the incorporation of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of HIV and HSV-1) into RBC, its delivery to macrophages and its activity against HIV or HSV-1. Loading of PMEA in artificially aged opsonized RBC affords significant levels of intracellular PMEA. RBC metabolize PMEA to its active congener PMEA-diphosphate, although with low efficiency. Exposure of macrophages to RBC-encapsulated PMEA inhibits the replication of both HIV and HSV-1 (about 90% inhibition at the highest RBC:macrophages ratios) even if RBC were removed before virus challenge. By contrast, the antiviral activity of free PMEA removed before virus challenge was irrelevant at concentrations up to 150-fold higher than the 50% effective concentration (EC50). Finally, the antiviral effect of RBC-encapsulated PMEA correlates with PMEA levels in macrophages about 500-fold higher than those achieved by free PMEA (at concentrations 10-fold higher than the EC50). The efficacy of RBC-mediated delivery to macrophages of PMEA (and perhaps of compounds with shorter intracellular half-lives) warrants further studies in infectious diseases involving phagocytizing cells as main targets of the pathogen.

HIV infection in macrophage: role of long-lived cells and related therapeutical strategies.

Therapeutical strategies aimed to the maximal inhibition (if not the eradication) of infection by human immunodeficiency virus should take into account the issue of the viral reservoir in the body. Recent data clearly show that latently infected lymphocytes represent a minimal part of the viral reservoir, while the majority of these cells are macrophages (variably differentiated) scattered in the tissues and lymph nodes. Immunologically-sequestred areas, such as the central nervous system, are particularly relevant in view of the different concentrations of antiviral drugs achieved in the organs. Thus, a careful analysis of the distribution of antiviral drugs, and the assessment of their activity in cells of macrophage lineage, represent key factors in the development of therapeutical strategies aimed to the "cure" of infectious patients.

Potent inhibition of human immunodeficiency virus and herpes simplex virus type 1 by 9-(2-phosphonylmethoxyethyl)adenine in primary macrophages is determined by drug metabolism, nucleotide pools, and cytokines.

The efficacy of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against the replication of human immunodeficiency virus (HIV) and herpes simplex virus type 1 (HSV-1) and its cellular metabolism were investigated in human primary macrophages from seronegative donors. PMEA potently inhibited the replication of both HIV and HSV-1 in macrophages, with similar EC50 values (0.025 and 0.032 microM, respectively), whereas the EC50 values of PMEA in lymphocytic C8166 cells and fibroblastoid Vero cells were 150-200-fold higher (3.5 and 7.9 microM, respectively). Granulocyte/macrophage colony-stimulating factor and macrophage colony-stimulating factor, two cytokine enhancers of the replication of HIV (and HSV-1), decreased the activity of PMEA against both viruses, yet EC50 values were still lower than in lymphocytes and fibroblasts. Thus, the selectivity index of PMEA in macrophages was > 2 orders of magnitude higher than that in lymphocytes and fibroblasts and still > 1 log higher under conditions of enhancement of virus replication in macrophages. The intracellular levels of 2'-deoxyadenosine-5'-triphosphate, the natural competitor of PMEA-diphosphate at the level of viral DNA polymerase (either RNA or DNA dependent), were 5-12-fold lower in macrophages than in other cells. Furthermore, intracellular concentrations of PMEA-diphosphate (the active metabolite of PMEA) were unusually much higher in macrophages (with or without cytokines) than in lymphocytes and fibroblasts. Consequently, the ratio of PMEA-diphosphate to 2'-deoxyadenosine-5'-triphosphate in monocytes/macrophages was approximately 2 orders of magnitude higher in macrophages than in the other cells and correlated closely with the pronounced antiviral potency of PMEA. The dual potent activity of PMEA against HIV and HSV-1 stresses the importance of clinical trials to assess the role of this drug in the therapy of HIV-related disease.

An ion-pairing high-performance liquid chromatographic method for the direct simultaneous determination of nucleotides, deoxynucleotides, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts.

An ion-pairing high-performance liquid chromatographic method for the direct and simultaneous determination of nucleotides, deoxynucleotides, cAMP, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts is presented. By using an Alltima C-18, 250 x 4.6-mm, 5-microns particle size column, a high resolution of 38 acid-soluble compounds, including ATP, GTP, dTTP, CTP, UTP, ADP, GDP, dTDP, CDP, UDP, dATP, dGTP, dCTP, dUTP, dADP, dGDP, dCDP, dUDP, and cAMP, is obtained. Elution is performed with a step gradient from buffer A (consisting of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4, 0.25% methanol, pH 7.00) to buffer B (consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4, 30% methanol, pH 5.50). Perchloric acid extracts of resting and phytohemagglutinin-stimulated human lymphocytes were analyzed. Data indicate that this chromatographic method offers, for the first time to the best of our knowledge, the possibility of simultaneously determining di- and triphosphate nucleosides and their corresponding deoxynucleosides without any chemical manipulation of samples except for perchloric acid deproteinization. Hence, the present HPLC assay minimizes the risks of modification or loss of metabolite concentration and allows one to obtain, with a single chromatographic run, the complete pattern of those metabolites which are known to be involved in energy metabolism and in DNA and RNA synthesis, resulting therefore of great advantage in cell biology studies.

Macrophage colony-stimulating factor enhances the susceptibility of macrophages to infection by human immunodeficiency virus and reduces the activity of compounds that inhibit virus binding.

The effects of macrophage colony-stimulating factor (M-CSF) on CD4 receptor expression, susceptibility to human immunodeficiency virus type 1 (HIV) infection, and anti-HIV activity of dextran sulfate and soluble-CD4 were studied in cultured, human primary macrophages. M-CSF stimulated macrophage cells to express the CD4 receptor, and this resulted in an increase of both the number of CD4+ cells and the density of the receptor on the cell surface. M-CSF also significantly enhanced the susceptibility of macrophage cells to HIV infection. Interestingly, the anti-HIV activity of dextran sulfate and soluble-CD4 (two compounds that interfere with HIV-CD4 binding with different mechanisms) was reduced 100-fold and fivefold, respectively, in M-CSF-treated macrophages. Human blood concentrations of M-CSF are reported to be similar to those used in this work (1,000 U/mL); thus, it is conceivable that also in vivo this cytokine may modify the susceptibility of macrophages to HIV and the ability of dextran sulfate and soluble CD4 to inhibit HIV replication. These results suggest that the in vitro study in M-CSF-treated macrophages of promising drugs inhibitors of HIV-CD4 binding could provide further insights into the potential efficacy of these compounds in patients.

9-(2-Phosphonylmethoxyethyl) adenine increases the survival of influenza virus-infected mice by an enhancement of the immune system.

PMEA (9-(2-phosphonylmethoxyethyl)adenine) is a potent inhibitor of DNA viruses and retroviruses able to enhance natural immune functions such as natural killer cell activity and interferon production. The results reported in this paper show that the treatment with PMEA significatively decreased the mortality of mice challenged with influenza A/PR8 virus (an RNA virus, non sensitive to the antiviral effect of PMEA) compared to untreated, infected controls (median survival 8.64 days and 7.61 days, respectively), and reduced lung weight and consolidation (two surrogate markers of virus infection). Furthermore, virus titer obtained from lung homogenates was substantially decreased in PMEA-treated mice compared to controls. Finally, enhancement of natural killer cell activity was achieved in PMEA-treated A/PR8-infected mice compared to A/PR8-infected controls. Overall, results suggest that PMEA decreases the influenza virus-related mortality and morbidity through the enhancement of some immune functions, and that this effect might be additive or even synergystic with the direct inhibitory effect of DNA viruses or retroviruses induced by PMEA itself. This supports the importance of evaluating this drug in patients with diseases related to herpesviruses or to human immunodeficiency virus.

In vitro activity of inhibitors of late stages of the replication of HIV in chronically infected macrophages.

Because of the importance of macrophages in the pathogenesis of the disease caused by HIV, we investigated the efficacy of various anti-HIV drugs in human primary macrophages acutely or chronically infected by this virus. The results obtained for acutely infected macrophages show that dideoxynucleosides (AZT, ddI, and ddC), interferon-alpha and -gamma, mismatched double-stranded RNA, Tat inhibitor, phosphorothioate antisense, and inhibitors of HIV protease, all significantly inhibit virus replication at concentrations far below those toxic for the cells. However, in macrophages in which proviral DNA is already integrated (chronically infected macrophages), only the three inhibitors of HIV protease induced significant virus inhibition at concentrations 100 or more times higher than those effective in acutely infected macrophages. Treatment of macrophages with macrophage colony-stimulating factor does not affect the anti-HIV efficacy of protease inhibitors. These results suggest that therapeutic strategies with activity for macrophages, including inhibitors of HIV protease, are worth pursuing in patients with HIV infection.

Enhancement of natural killer activity and interferon induction by different acyclic nucleoside phosphonates.

Acyclic nucleoside phosphonate (ANP) analogues are a class of compounds with potent activity against herpesviruses and/or retroviruses. Our preliminary experiments have shown that 9-(2-phosphonylmethoxyethyl)adenine (PMEA), a prototype of the ANP family, enhances some parameters of natural immunity. In this paper we have evaluated the effect of different schedules of administration of PMEA and other ANP analogues of clinical interest upon natural killer (NK) activity and interferon (IFN) production in a mouse model. The results show that PMEA significantly enhances NK activity and interferon production. Other ANP analogues tested in our system, i.e., 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA), similarly induced enhancement of natural immunity. The immunomodulating effect of PMEA was even more pronounced with a single administration compared to repeated administrations of the drug. Dose-dependent enhancement of NK activity and IFN production could also be demonstrated during chronic administration of PMEA (more resembling to what will be the schedule of administration of this drug in patients). Overall, the data here presented suggest that the enhancement of some natural immune functions induced by ANP analogues may add to the direct antiviral activity of these drugs against retroviruses and herpesviruses, and thus may be able to increase the host resistance against viral infections.