Efavirenz does not alter JNK phosphorylation status in platelets, but does induce activation of GSK3β.

<p>Washed human platelets were treated as indicated for 1 h. Graphs indicate fold change in phosphorylation as compared to non-treated (NT) and determined via densitometry of immunoblots (representative blots shown above corresponding treatments), with phospho -JNK or -GSK3β quantification determined as a percentage of the total amount of each protein for each sample. (A) EFV does not induce activation of JNK, while thrombin treatment, as a positive control, induced phosphorylation of this kinase, indicating activation. This was partially reversed when treated in the presence of increasing amounts of the JNK inhibitor SP600125 (SP). Values were compared using an one-way ANOVA followed by Bonferroni’s test for multiple comparisons, which indicated statistical significance as *p<0.05 and **p<0.01 compared to all other conditions. N.S. indicates not significant; D indicates DMSO as a vehicle control. (B) EFV treatment resulted in dephosphorylation of GSK3β at the inhibitory phosphorylation site (Ser9), indicating activation of this molecule in platelets. One-way ANOVA followed by Bonferroni’s test for multiple comparisons indicated statistical significance as *p<0.05, **p<0.01, and ***p<0.001.</p

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) induce sCD40L release from washed human platelets.

<p>(A) Platelets isolated from healthy donors were treated as indicated and analyzed via ELISA specific for sCD40L. Efavirenz (EFV), but not the protease inhibitors lopinavir and ritonavir (LOPV+RITV), induce sCD40L release, indicating that EFV directly activates platelets. (B) EFV induces sCD40L release in a dose dependent manner in washed human platelets, with significance achieved at physiologically relevant concentrations. (C) The NNRTI nevirapine (NEVP) also induces the release of sCD40L directly from isolated human platelets. In panels A – C values were compared via one-way ANOVA followed by Bonferroni’s test for multiple comparisons, which indicated statistical significance as *p<0.05, **p<0.01, and ***p<0.001.</p

Efavirenz, a non-nucleoside reverse transcriptase inhibitor (NNRTI), results in accumulation of sCD40L in HIV infected individuals.

<p>Plasma sCD40L levels were analyzed in HIV infected individuals receiving combination antiretroviral therapy including NRTIs (abacavir, ABCV, and lamivudine, LAMV; Control, n = 12) and either an NNRTI (efavirenz, EFV; n = 13) or protease inhibitor cocktail (lopinavir, LOPV, and ritonavir, RITV; n = 10). The values were compared via one-way ANOVA followed by Bonferroni’s test for multiple comparisons, which indicated statistical significance as **p<0.01.</p

CD40L is required for Tat-induced BBB permeability.

<p>(<b>A</b>) Wild-type C57BL/6 (WT) or CD40L deficient (CD40L KO) mice were injected retro-orbitally with HIV Tat (1 µg/g body weight; n = 6 per group), while control mice were injected with saline. Sodium fluorescein (NaF) analysis revealed that the Tat-induced increase in BBB permeability is dependent on CD40L. The values shown here represent fold increase in the ratio of brain versus plasma concentrations of NaF. (<b>B</b>) Complete blood counts reveal that both WT and CD40L KO animals demonstrate a Tat-induced drop in platelet count by 24 h post-treatment. For panels (A) and (B) statistical significance was determined via one-way ANOVA followed by Bonferroni’s test for multiple comparisons and indicated in the figure as *p<0.05. (<b>C</b>) Verification that CD40L expression is absent in CD40L KO animals. Representative results obtained from reverse transcription-PCR of spleen homogenates using primers specific for CD40L and GAPDH. (<b>D</b>) Recombinant mouse sCD40L (rsCD40L; 0.2 µg/g body weight) was injected intraperitoneally into WT animals (n = 4). Twelve hours post-treatment NaF assays were performed and it was revealed that rsCD40L induced similar BBB permeability as treatment with Tat alone. An unpaired t-test indicated that p = 0.0531. sCD40L ELISA confirmed that there was a significant increase in circulating sCD40L following treatment with rsCD40L (right panel), and an unpaired t-test determined statistical significance as ***p<0.001.</p

Tat increases the number of rolling and adhered leukocytes to the brain microvasculature.

<p>(<b>A</b>) Representative images of cortical two-photon time-lapse videos in wild-type C57BL/6 (WT) or CD40L deficient (CD40L KO) mice (n = 3). Tat (1 µg/g body weight) was injected retro-orbitally 24 h prior to creation of cortical window and subsequent imaging. The cerebral blood vessels were fluorescently labeled with Texas Red Dextran that was injected into the femoral vein prior to acquisition. Leukocyte rolling and adhesion was visualized using Alexa Fluor 488-conjugated antibody against the granulocyte antigen 1 (Gr1) that was injected in the same manner. Tat treatment in WT, but not CD40L KO, mice induced an increase in the number of rolling (arrows) and adhered (circles) Gr1 positive cells. (<b>B</b>) Quantitation of Gr1 positive cells rolling on or adhered to vessels in the two-photon time-lapse videos. (<b>C</b>) WT or CD40L KO animals were treated as in (A) and whole blood was collected via cardiac exsanguination. Subsequent flow cytometric analysis of monocytes using the same fluorescently labeled anti-Gr1 antibody (left panel shows representative Gr1 positive gating) detected an equal number of Gr1 positive, inflammatory monocytes in both WT and CD40L KO animals. (<b>D</b>) Samples described in (C) were also analyzed using fluorescently labeled anti-CCR-2 to monitor chemokine receptor expression in response to Tat in both WT and CD40L KO mice. (B–D) Values in each panel were compared using one-way ANOVA followed by Bonferroni’s test for multiple comparisons with statistical significance defined as *p<0.05 and ***p<0.001.</p

HIV Tat activates platelets <i>in vivo.</i>

<p>(<b>A</b>) Plasma concentrations of platelet factor 4 (PF4) and soluble CD40L (sCD40L) in wild-type C57BL/6 (WT) mice were measured via ELISA. Upon injection with HIV Tat (100 ng/g body weight; n = 5), there is a significant increase in the level of each of these platelet-derived mediators in the plasma after 1 h, indicating that Tat stimulates platelets <i>in vivo</i>. Samples were compared using an unpaired t-test with statistical significance as **p<0.01 and ***p<0.001. (<b>B</b>) Tat significantly decreased the time to clot in WT mice that had been nicked in the tail, indicating platelet activation. Tat that had been heat inactivated (H.I. Tat) prior to treatment did not significantly reduce bleeding time as compared to saline treatment. Samples were compared via one-way ANOVA followed by Bonferroni’s test for multiple comparisons, which indicated statistical significance as **p<0.01 for the Tat treated animals compared to both saline and H.I. Tat.</p

Tat increases blood brain barrier (BBB) permeability.

<p>(<b>A</b>) Wild-type C57BL/6 (WT) mice were treated with Tat (1 µg/g body weight; n = 6) for 24 h. The fluorescent tracer sodium fluorescein (NaF) was then used to assess BBB permeability. HIV Tat, but not heat inactivated Tat (H.I. Tat), significantly increased BBB permeability as compared to saline treated animals. The values are presented as fold increase in the ratio of brain versus plasma concentrations of NaF. (<b>B</b>) The Tat-induced increase in vascular permeability was limited to the BBB in our model since there was no increase in fluorescence in other tissues, such as the spleen or kidney, in the same WT animals. Groups were compared in both (A) and (B) using one-way ANOVA followed by Bonferroni’s test for multiple comparisons with statistical significance indicated as *p<0.05 or n.s. as not significant. (<b>C</b>) Complete blood counts indicated that there was a Tat-induced drop in platelet count by 24 h, indicative of activation followed by consumption of these cells. Groups were compared individually using a paired t-test with significance indicated as *p<0.05.</p

Platelet-derived sCD40L is contributing to Tat-induced BBB permeability.

<p>(<b>A</b>) Complete blood counts verified efficient platelet depletion in wild-type C57BL/6 (WT) animals (n = 6) 24 h following treatment with antibodies for either platelet depletion or control, non-immune rat immunoglobulin (Control IgG; 0.5 µg/g body weight of either antibody). Tat treatment (1 µg/g body weight) also led to a significant loss of platelets in control antibody treated, but not platelet depleted, animals (at 48 h post-depletion, 24 h following Tat treatment). Legend indicates time of complete blood counts post antibody injection. (<b>B</b>) Sodium fluorescein analysis of animals treated in (A) revealed that platelets are required for the Tat-induced increase in BBB permeability. (<b>C</b>) Whole blood was collected via cardiac exsanguination from animals treated as in (A) and ELISA specific for sCD40L was performed on platelet poor plasma samples. As expected, ELISA analysis revealed a Tat-induced increase in sCD40L in animals that had been treated with control antibodies; however in animals that had been depleted of platelets, concentrations were lower then saline treated control animals, indicating that platelets are the major source of circulating sCD40L. In panels (A−C) samples were compared via one-way ANOVA followed by Bonferonni’s test for multiple comparisons with statistical significance indicated as *p<0.05, **p<0.01, and ***p<0.001.</p

Short-Term Pulmonary Toxicity Assessment of Pre- and Post-incinerated Organomodified Nanoclay in Mice

Organomodified nanoclays (ONCs) are increasingly used as filler materials to improve nanocomposite strength, wettability, flammability, and durability. However, pulmonary risks associated with exposure along their chemical lifecycle are unknown. This study’s objective was to compare pre- and post-incinerated forms of uncoated and organomodified nanoclays for potential pulmonary inflammation, toxicity, and systemic blood response. Mice were exposed <i>via</i> aspiration to low (30 μg) and high (300 μg) doses of preincinerated uncoated montmorillonite nanoclay (CloisNa), ONC (Clois30B), their respective incinerated forms (I-CloisNa and I-Clois30B), and crystalline silica (CS). Lung and blood tissues were collected at days 1, 7, and 28 to compare toxicity and inflammation indices. Well-dispersed CloisNa caused a robust inflammatory response characterized by neutrophils, macrophages, and particle-laden granulomas. Alternatively, Clois30B, I-Clois30B, and CS high-dose exposures elicited a low grade, persistent inflammatory response. High-dose Clois30B exposure exhibited moderate increases in lung damage markers and a delayed macrophage recruitment cytokine signature peaking at day 7 followed by a fibrotic tissue signature at day 28, similar to CloisNa. I-CloisNa exhibited acute, transient inflammation with quick recovery. Conversely, high-dose I-Clois30B caused a weak initial inflammatory signal but showed comparable pro-inflammatory signaling to CS at day 28. The data demonstrate that ONC pulmonary toxicity and inflammatory potential relies on coating presence and incineration status in that coated and incinerated nanoclay exhibited less inflammation and granuloma formation than pristine montmorillonite. High doses of both pre- and post-incinerated ONC, with different surface morphologies, may harbor potential pulmonary health hazards over long-term occupational exposures

Short-Term Pulmonary Toxicity Assessment of Pre- and Post-incinerated Organomodified Nanoclay in Mice

Organomodified nanoclays (ONCs) are increasingly used as filler materials to improve nanocomposite strength, wettability, flammability, and durability. However, pulmonary risks associated with exposure along their chemical lifecycle are unknown. This study’s objective was to compare pre- and post-incinerated forms of uncoated and organomodified nanoclays for potential pulmonary inflammation, toxicity, and systemic blood response. Mice were exposed <i>via</i> aspiration to low (30 μg) and high (300 μg) doses of preincinerated uncoated montmorillonite nanoclay (CloisNa), ONC (Clois30B), their respective incinerated forms (I-CloisNa and I-Clois30B), and crystalline silica (CS). Lung and blood tissues were collected at days 1, 7, and 28 to compare toxicity and inflammation indices. Well-dispersed CloisNa caused a robust inflammatory response characterized by neutrophils, macrophages, and particle-laden granulomas. Alternatively, Clois30B, I-Clois30B, and CS high-dose exposures elicited a low grade, persistent inflammatory response. High-dose Clois30B exposure exhibited moderate increases in lung damage markers and a delayed macrophage recruitment cytokine signature peaking at day 7 followed by a fibrotic tissue signature at day 28, similar to CloisNa. I-CloisNa exhibited acute, transient inflammation with quick recovery. Conversely, high-dose I-Clois30B caused a weak initial inflammatory signal but showed comparable pro-inflammatory signaling to CS at day 28. The data demonstrate that ONC pulmonary toxicity and inflammatory potential relies on coating presence and incineration status in that coated and incinerated nanoclay exhibited less inflammation and granuloma formation than pristine montmorillonite. High doses of both pre- and post-incinerated ONC, with different surface morphologies, may harbor potential pulmonary health hazards over long-term occupational exposures