Glutamate-stimulated peroxynitrite production in a brain-derived endothelial cell line is dependent on N-methyl-D-aspartate (NMDA) receptor activation.

There is accumulating and convincing evidence indicating a role for glutamate in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses neurological symptoms and prevents blood-brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during EAE remain unclear. Glutamate triggers the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite (ONOO(-)). Recent studies have implicated ONOO(-) in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown via the actions of ONOO(-). The present investigation examined glutamate-induced ONOO(-) formation in the b.End3 brain-derived endothelial cell line. b.End3 cells were incubated with a concentration range of glutamate and ONOO(-) production was assessed over time. Results showed a concentration- and time-dependent increase in ONOO(-) levels in glutamate-treated cells that were suppressed by selective and non-selective inhibitors of ONOO(-)-mediated reactions. Specific activation of b.End3-associated NMDA receptors also resulted in a concentration-dependent increase in ONOO(-) production. The ability of b.End3 cells to respond to the presence of glutamate was confirmed through the detection of NMDA receptor immnuoreactivity in cell extracts. In addition, the use of the NMDA receptor antagonists MK-801 and memantine reduced glutamate-mediated ONOO(-) generation from b.End3 cells. The data reinforce the important relationship between glutamate and the NMDA receptor, positioned at neurovascular sites, which may be of particular relevance to the pathogenesis of demyelinating disease.

Altered circadian relationship between serum nitric oxide, carbon dioxide, and uric acid in multiple sclerosis.

The free radical nitric oxide (NO*) is involved in a variety of diverse biological processes from acting as a vasodilator in the cardiovascular system to being the rate-limiting component in the production of peroxynitrite (ONOO-), a contributor to neurodegenerative disorders such as multiple sclerosis (MS). Uric acid (UA), the end product of purine metabolism in humans and a selective inhibitor of toxic reactions attributed to radicals formed by the interaction of ONOO- and CO2, is generally low in MS patients. We investigated the relationship between serum ONOO-, CO2, and UA in MS patients and normal controls by comparing the circadian characteristics of the NO* metabolites nitrite/ nitrate (NO), CO2, and UA. In this preliminary study, we found the functional relationship ascribed to the circadian timing of the peak and trough levels of NO, CO2, and UA in healthy subjects to be clearly altered in MS patients. These findings suggest that alterations in the temporal relationship between the 24h pattern in serum ONOO- formation and UA may either contribute to or reflect the disease processes in MS.

The central nervous system inflammatory response to neurotropic virus infection is peroxynitrite dependent.

We have recently demonstrated that increased blood-CNS barrier permeability and CNS inflammation in a conventional mouse model of experimental allergic encephalomyelitis are dependent upon the production of peroxynitrite (ONOO(-)), a product of the free radicals NO* and superoxide (O2*(-)). To determine whether this is a reflection of the physiological contribution of ONOO(-) to an immune response against a neurotropic pathogen, we have assessed the effects on adult rats acutely infected with Borna disease virus (BDV) of administration of uric acid (UA), an inhibitor of select chemical reactions associated with ONOO(-). The pathogenesis of acute Borna disease in immunocompetent adult rats results from the immune response to the neurotropic BDV, rather than the direct effects of BDV infection of neurons. An important stage in the BDV-specific neuroimmune response is the invasion of inflammatory cells into the CNS. UA treatment inhibited the onset of clinical disease, and prevented the elevated blood-brain barrier permeability as well as CNS inflammation seen in control-treated BDV-infected rats. The replication and spread of BDV in the CNS were unchanged by the administration of UA, and only minimal effects on the immune response to BDV Ags were observed. These results indicate that the CNS inflammatory response to neurotropic virus infection is likely to be dependent upon the activity of ONOO(-) or its products on the blood-brain barrier.

Effect of mercaptoethylguanidine scavengers of peroxynitrite on the development of experimental allergic encephalomyelitis in PLSJL mice.

Peroxynitrite has been implicated in the pathogenesis of multiple sclerosis and its animal counterpart experimental allergic encephalomyelitis (EAE). Here we have examined the effects of the novel peroxynitrite scavengers, mercaptoethylguanidine (MEG) and guanidinoethyldisulphide (GED), on the development of EAE. Both MEG and GED delayed EAE onset and decreased the number of animals displaying disease signs. However, when EAE developed, its severity was not significantly abrogated by drug administration. These results suggest that while MEG and GED protect against the induction phase of EAE, they do not prevent disease progression. This may be due to the inability of MEG and GED to efficiently scavenge peroxynitrite or result from their capacity to inhibit inducible nitric oxide synthase. Therefore, the development of more potent and selective scavengers of peroxynitrite is necessary for use in EAE.

Role of poly(ADP-ribose) synthetase activation in the development of experimental allergic encephalomyelitis.

Peroxynitrite formation has been demonstrated during experimental allergic encephalomyelitis (EAE). Furthermore, peroxynitrite has been identified as an activator of poly(ADP-ribose) synthetase (PARS), an enzyme implicated in neurotoxicity. In the current study, we examined the role of PARS activation in the development of EAE. Administration of the PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) delayed the onset of EAE and reduced the incidence and severity of disease signs. Moreover, drug treatment lowered iNOS activity and decreased cell infiltration in cervical spinal tissues from EAE-sensitized animals. To conclude, the results of the present investigation suggest that PARS activity may contribute to the pathogenesis of EAE.

The role of uric acid in protection against peroxynitrite-mediated pathology.

Peroxynitrite, the product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of inflammatory CNS disorders. Uric acid, an effective scavenger of peroxynitrite, is a purine metabolite present at high levels in the serum of hominoids relative to lower-order animals due to the functional deletion of urate oxidase. Raising the normally low levels of uric acid in mice is therapeutic for experimental allergic encephalomyelitis, an animal model of multiple sclerosis. This therapeutic activity of uric acid is associated with the inhibition of peroxynitrite-induced tissue damage, blood-CNS barrier permeability changes, and CNS inflammation. Based on these findings we have concluded that peroxynitrite has an important role in promoting enhanced vascular permeability and inflammatory cell extravasation. We hypothesize that higher uric acid levels in hominoids evolved to protect against this process.

The peroxynitrite scavenger uric acid prevents inflammatory cell invasion into the central nervous system in experimental allergic encephalomyelitis through maintenance of blood-central nervous system barrier integrity.

Uric acid (UA), a product of purine metabolism, is a known scavenger of peroxynitrite (ONOO(-)), which has been implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis (EAE). To determine whether the known therapeutic action of UA in EAE is mediated through its capacity to inactivate ONOO(-) or some other immunoregulatory phenomenon, the effects of UA on Ag presentation, T cell reactivity, Ab production, and evidence of CNS inflammation were assessed. The inclusion of physiological levels of UA in culture effectively inhibited ONOO(-)-mediated oxidation as well as tyrosine nitration, which has been associated with damage in EAE and multiple sclerosis, but had no inhibitory effect on the T cell-proliferative response to myelin basic protein (MBP) or on APC function. In addition, UA treatment was found to have no notable effect on the development of the immune response to MBP in vivo, as measured by the production of MBP-specific Ab and the induction of MBP-specific T cells. The appearance of cells expressing mRNA for inducible NO synthase in the circulation of MBP-immunized mice was also unaffected by UA treatment. However, in UA-treated animals, the blood-CNS barrier breakdown normally associated with EAE did not occur, and inducible NO synthase-positive cells most often failed to reach CNS tissue. These findings are consistent with the notion that UA is therapeutic in EAE by inactivating ONOO(-), or a related molecule, which is produced by activated monocytes and contributes to both enhanced blood-CNS barrier permeability as well as CNS tissue pathology.

Protection of myelin basic protein immunized mice from free-radical mediated inflammatory cell invasion of the central nervous system by the natural peroxynitrite scavenger uric acid.

Peroxynitrite (ONOO(-)), the product of nitric oxide (NO(radical)) and superoxide (O(2)(-radical)), is believed to be a major contributor to immunotoxicity when produced by activated cells expressing inducible nitric oxide synthase (iNOS). Uric acid (UA) is a natural scavenger of ONOO(-) that is present at high levels in the sera of humans and other higher order primates relative to most lower mammals. We have previously shown that UA treatment is therapeutic in experimental allergic encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). In this study we have examined the effect of UA therapy on the dynamics of the appearance of iNOS-positive cells in central nervous system (CNS) tissue of mice subjected to the stimuli that cause EAE. The results indicate that UA prevents activated monocytes from entering CNS tissue where they may contribute to the pathogenesis of MS and other CNS diseases.

Language development and assessment in children with human immunodeficiency virus: 3 to 6 years.

Young children infected or exposed, or both to the human immunodeficiency virus (HIV) present with a variety of speech, language, and communication problems. The purpose of this article is to provide an overview of the impact that HIV has on young children from 3 to 6 years of age. Issues concerning medically related problems are discussed, along with assessment criteria and descriptions of communication disorders among HIV-infected and -exposed children.

Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis.

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.

Effects of nicaraven on nitric oxide-related pathways and in shock and inflammation.

Expression of the inducible isoform of nitric oxide (NO) synthase, and the formation of peroxynitrite from NO and superoxide are responsible for some of the pathophysiological alterations seen during reperfusion injury and in various inflammatory conditions. Some of the effects of peroxynitrite are related to DNA single-strand breakage, and activation of poly (ADP-ribose) synthetase. Here we investigated the effect of nicaraven (2(R,S)-1,2-bis(nicotinamido)propane), a known hydroxyl radical scavenger compound and neuroprotective agent, on several NO- and peroxynitrite related pathways in vitro, and in shock and inflammation in vivo. Nicaraven, at 10 microM-10 mM, failed to inhibit the peroxynitrite-induced oxidation of dihydrorhodamine 123, indicating that the agent does not act as a scavenger of peroxynitrite. In RAW murine macrophages stimulated with peroxynitrite, nicaraven caused a dose-dependent, slight inhibition of poly (ADP-ribose) synthetase activation, possibly due to a direct inhibitory effect on the catalytic activity of poly (ADP-ribose) synthetase. Nicaraven partially protected against the peroxynitrite-induced suppression of mitochondrial respiration in RAW macrophages and caused a slight, dose-dependent inhibition of nitrite production in RAW macrophages stimulated with bacterial lipopolysaccharide. We next investigated the effect of nicaraven treatment in a variety of models of inflammation and reperfusion injury. Nicaraven (at 10-100 microg/paw) exerted significant protective effects in the carrageenan-induced paw edema model and (at 100 mg/kg i.v.) reduced neutrophil infiltration and histological damage in splanchnic artery occlusion-reperfusion injury. However, nicaraven failed to alter the course of hemorrhagic and endotoxic shock and arthritis in rodent models. The current data indicate the limited role of hydroxyl radicals in the pathogenesis of the inflammatory conditions tested.

L-arginine modifies free radical production and the development of experimental allergic encephalomyelitis.

OBJECTIVES AND DESIGN: The present investigation examines the effects of increasing central nervous system (CNS) levels of nitric oxide (NO), via the administration of L-arginine, on the development of experimental allergic encephalomyelitis (EAE). SUBJECTS: EAE was induced in male Lewis rats (200-250 g). TREATMENT: Normal rats were orally dosed with L-arginine (300 mg/kg body weight) once daily for 12 days. EAE-sensitised animals received L-arginine (300 mg/kg body weight) once daily from day 1 to 12 post-inoculation. METHODS: Neurological and histological development of EAE were assessed. In addition, CNS cytosol levels of nitrite, superoxide and hydrogen peroxide were measured. Results were analysed using the Mann Whitney U-test and Chi-squared test. RESULTS: L-arginine administration significantly delayed disease onset (p < 0.05) and reduced the severity of neurological (p < 0.05) and histological (p < 0.001) signs of EAE. Treatment with L-arginine caused a significant elevation in CNS nitrite concentrations (p < 0.05) which in EAE-sensitised animals was associated with a concomitant and dramatic decrease in superoxide (p < 0.05) and hydrogen peroxide (p < 0.05) levels. CONCLUSIONS: The results suggest that NO may act as a protective molecule during the development of EAE possibly via the modulation of oxidant-mediated neuroinflammation.

Requirement of intracellular calcium mobilization for peroxynitrite-induced poly(ADP-ribose) synthetase activation and cytotoxicity.

Peroxynitrite is a cytotoxic oxidant produced during shock, ischemia reperfusion, and inflammation. The cellular events mediating the cytotoxic effect of peroxynitrite include activation of poly(ADP-ribose) synthetase, inhibition of mitochondrial respiration, and activation of caspase-3. The aim of the present study was to investigate the role of intracellular calcium mobilization in the necrotic and apoptotic cell death induced by peroxynitrite. Peroxynitrite, in a low, pathophysiologically relevant concentration (20 microM), induces rapid (1 to 3 min) Ca(2+) mobilization in thymocytes. Inhibition of this early calcium signaling by cell-permeable Ca(2+) chelators [EGTA-acetoxymethyl ester (AM), 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N , N',N'-tetraacetic acid-tetra-AM] abolished cytotoxicity as measured by propidium iodide uptake. Intracellular Ca(2+) chelators also inhibited DNA single-strand breakage and activation of poly(ADP-ribose) synthase (PARS), which is a major mediator of cell necrosis in the current model. Intracellular Ca(2+) chelators also protected PARS-deficient thymocytes from peroxynitrite cytotoxicity, providing evidence for a PARS-independent, Ca(2+)-dependent cytotoxic pathway. Chelation of intracellular Ca(2+) blocked the peroxynitrite-induced decrease of mitochondrial membrane potential, secondary superoxide production, and mitochondrial membrane damage. Peroxynitrite-induced internucleosomal DNA cleavage was increased on BAPTA-AM pretreatment in the wild-type cells but decreased in the PARS-deficient cells. Two other apoptotic parameters (phosphatidylserine exposure and caspase 3 activation) were inhibited by BAPTA-AM in both the wild-type and the PARS-deficient thymocytes. Our findings provide evidence for the pivotal role of an early Ca(2+) signaling in peroxynitrite cytotoxicity.

Peroxynitrite production and activation of poly (adenosine diphosphate-ribose) synthetase in spinal cord injury.

Peroxynitrite formation and the subsequent activation of the nuclear enzyme, poly (adenosine diphosphate [ADP]-ribose) synthetase (PARS), has been implicated in the pathogenesis of several neurodegenerative disorders. Here, we demonstrate that nitrotyrosine, an indicator of peroxynitrite generation, and poly (ADP) ribose, a marker of PARS activation, were selectively localized within tissues from spinal cord-injured rats. Our data implicate a role for peroxynitrite production and PARS activation in the development of spinal cord trauma.

Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation.

The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross-linking of T-cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno-histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10 microM) of peroxynitrite led to apoptosis, whereas higher concentrations (50 microM) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP-ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite-induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy-conserving step, enabling the cell to complete the process of apoptosis.

Suppression of macrophage inflammatory protein (MIP)-1alpha production and collagen-induced arthritis by adenosine receptor agonists.

1. Ligands of the various adenosine receptor subtypes modulate the production of pro-and anti-inflammatory cytokines. Here we evaluated the effect of adenosine and various ligands of the adenosine receptor subtypes (A1, A2, A3) on the chemokine macrophage inflammatory protein (MIP) 1alpha production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A3 adenosine receptor agonist inhibits MIP-1alpha production and affects the course of inflammation in collagen-induced arthritis. 2. In the cultured macrophages, the A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), and, less potently, the A2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS; 1-200 micro) dose-dependently suppressed the production of MIP-1alpha. The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1-200 microM) was ineffective, and adenosine was a weak inhibitor. The inhibition of MIP-1alpha production by the A3 and A2 agonist was associated with suppression of its steady-state mRNA levels. 3. Based on the in vitro data, we concluded that activation of A3, and to a lesser extent A2 adenosine receptors suppresses MIP-1alpha expression. Since IB-MECA was the most potent inhibitor of MIP-1alpha expression, we next investigated whether it affects the production of other pro-inflammatory mediators. We observed that IB-MECA (1-300 microM) inhibited, in a dose-dependent manner, the production of IL-12, IL-6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages. 4. Since MIP-alpha is a chemokine which enhances neutrophil recruitment into inflammatory sites, we investigated whether the A3 agonist IB-MECA affects the course of inflammation, MIP-alpha production and the degree of neutrophil recruitment in arthritis. In a model of collagen-induced arthritis in mice, IB-MECA (0.5 mg/kg/day) reduced the severity of joint inflammation. IB-MECA inhibited the formation of MIP-1alpha, IL-12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil infiltration. 5. We conclude that adenosine receptor agonists, most notably the A3 agonist IB-MECA suppress the production of MIP-alpha, and exert anti-inflammatory effects. Therefore, stimulation of adenosine receptor subtypes A3 and A2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic inflammatory disorders.

Protective effects of 5-iodo-6-amino-1,2-benzopyrone, an inhibitor of poly(ADP-ribose) synthetase against peroxynitrite-induced glial damage and stroke development.

Peroxynitrite triggers DNA single-strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). Activation of PARS depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation, resulting in cell necrosis. Here, we demonstrate that inhibition of PARS with the novel, potent PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) protects against peroxynitrite-induced cell death (as measured by measurement of mitochondrial respiration and release of lactate dehydrogenase) in C6 glioma cells in vitro, and in a murine stroke model in vivo. Inhibition of PARS with INH2BP may represent a novel approach for the experimental therapy of stroke.

Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase.

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS-/-) and corresponding wild-type animals (PARS+/+), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Exposure of embryonic fibroblasts from the PARS+/+ animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS+/+ cells against the suppression of mitochondrial respiration in response to peroxynitrite (50-100 microM). Similarly to PARS inhibition with INH2BP, the PARS-/- cells were protected against peroxynitrite-induced injury. The protection against cellular injury by PARS-/- phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS-/- phenotype reduced inducible nitric-oxide synthase (iNOS; EC 1.14.13.39) mRNA levels and inhibited production of NO in immunostimulated cells. INH2BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS-/- cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inflamed joints. Oral treatment with INH2BP (0.5 g/kg, daily), starting at the onset of arthritis (day 25), delayed the development of the clinical signs at days 26-35 and improved histological status in the knee and paw. Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protects against oxidant-induced cellular injury in vitro and exhibits anti-inflammatory effects in vivo.

Epidemiologic principles in studies of infectious disease outcomes: pediatric HIV as a model.

Epidemiologic principles have been employed in the investigation of AIDS since the early 1980s. Although such principles have demonstrated the difficulties in reporting the everchanging rates of incidence and prevalence, in addition to distributions of children with HIV, they have also established specific pieces of a multifaceted puzzle. Professionals interested in examining only a piece of the puzzle, such as a particular communication disorder, often are unable to see how it fits into the complete puzzle. This article presents several epidemiologic findings of pediatric HIV, including population distributions, a summary of modes of transmission, occurrence of opportunistic infections, and manifestations of the disease in child populations. It also discusses HIV-related speech, language, swallowing, and voice disorders, examining the complexities of quantification of risk for each piece within the pediatric HIV puzzle. The purpose is to broaden the perspective of professionals concerned with how these disorders fit within the overall puzzle of immunocompromised populations of children.