Therapeutic Efficacy and Immunological Response of CCL5 Antagonists in Models of Contact Skin Reaction

Skin-infiltrating T-cells play a predominant role in allergic and inflammatory skin diseases such as atopic dermatitis, psoriasis and allergic contact dermatitis. These T-cells are attracted by several chemotactic factors including the chemokine CCL5/RANTES, a CC chemokine inducing both the migration and activation of specific leukocyte subsets. CCL5 has been found to be associated with various cell-mediated hypersensitive disorders such as psoriasis, atopic dermatitis and irritant contact dermatitis. We have used two antagonists, the first, Met-CCL5, a dual CCR1/CCR5 antagonist and the second, a variant in which GAG binding is abrogated, 44AANA47-CCL5, which acts as a dominant negative inhibitor of CCL5. The antagonists were tested in two models of contact skin reaction. The first, irritant contact dermatitis (ICD) is a pathological non-specific inflammatory skin condition arising from the release of pro-inflammatory cytokines by keratinocytes in response to haptens, usually chemicals. The second, contact hypersensitivity (CHS) is a T-cell dependent model, mimicking in part the T-cell-mediated skin diseases such as psoriasis. In both models, the CCL5 antagonists showed therapeutic efficacy by reducing swelling by 50% as well as the reduction of soluble mediators in homogenates derived from challenged ears. These results demonstrate that blocking the receptor or the ligand are both effective strategies to inhibit skin inflammation

Preclinical evaluation of FLT190, a liver-directed AAV gene therapy for Fabry disease

Fabry disease is an X-linked lysosomal storage disorder caused by loss of alpha-galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of glycosphingolipids in multiple cells and tissues. FLT190, an investigational gene therapy, is currently being evaluated in a Phase 1/2 clinical trial in patients with Fabry disease (NCT04040049). FLT190 consists of a potent, synthetic capsid (AAVS3) containing an expression cassette with a codon-optimized human GLA cDNA under the control of a liver-specific promoter FRE1 (AAV2/S3-FRE1-GLAco). For mouse studies FLT190 genome was pseudotyped with AAV8 for efficient transduction. Preclinical studies in a murine model of Fabry disease (Gla-deficient mice), and non-human primates (NHPs) showed dose-dependent increases in plasma α-Gal A with steady-state observed 2 weeks following a single intravenous dose. In Fabry mice, AAV8-FLT190 treatment resulted in clearance of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) in plasma, urine, kidney, and heart; electron microscopy analyses confirmed reductions in storage inclusion bodies in kidney and heart. In NHPs, α-Gal A expression was consistent with the levels of hGLA mRNA in liver, and no FLT190-related toxicities or adverse events were observed. Taken together, these studies demonstrate preclinical proof-of-concept of liver-directed gene therapy with FLT190 for the treatment of Fabry disease

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Vascular endothelial growth factor (VEGF) and lovastatin suppress the inflammatory response to Plasmodium berghei infection and protect against experimental cerebral malaria

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, which is associated with high mortality and long-term cognitive impairment even when effective anti-parasitic treatment is administered. (1 , 2) Supportive therapy is needed to improve both morbidity and mortality associated with this condition. In an accompanying paper, we have demonstrated that in the Plasmodium berghei ANKA (PbA) rodent model, CM can be effectively prevented by a treatment combining sub-lethal doses of lipopolysaccharide S (LPS) and vascular endothelial growth factor (VEGF). Since LPS is not suitable for human therapy, we investigated whether lovastatin would represent a suitable substitute. This compound, widely used to lower cholesterol levels in plasma, shares with LPS the ability to elicit an anti-inflammatory response by activating the Nrf-2 gene, and when given to P. berghei-infected mice prevents to some extent the onset of CM. We show here that lovastatin- and VEGF-treated mice did not develop CM and showed few signs, if any, of endothelial damage and systemic inflammation. The combination treatment was much more effective than lovastatin and VEGF alone. Immunohistochemistry and gene expression analysis indicated that VEGF and LPS together overturned the two pathogenic mechanisms responsible for the development of CM: endothelial damage and disregulated activation of the inflammatory response. These findings provide the rationale for investigating the therapeutic potential of these compounds in human CM as well as in other inflammatory pathologies that respond poorly to steroid and non-steroid anti-inflammatory therapy

Therapeutic efficacy of [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 on ear swelling, using different mouse models of contact skin reaction.

<p><b>A</b>) represents [⁴⁴AANA⁴⁷]-CCL5 and <b>B</b>) Met-CCL5 therapeutic effect respectively on swelling in Oxazolone-induced CHS mouse model at 0.05–0.5–1 mg/kg dose via i.p. Dexamethasone 10 mg/kg sub-cute (s.c.) was used as reference compound. <b>C</b>) represents [⁴⁴AANA⁴⁷]-CCL5 and <b>D</b>) Met-CCL5 therapeutic effect respectively on swelling in CHS mouse model using DNFB as hapten at 0.05–0.5–1 mg/kg via i.p. Dexamethasone 0.5 mg/kg s.c. was used as reference compound. <b>E–F</b>) are showing therapeutic efficacy of [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 in ICD mouse model tested at 0.5–1–5 mg/kg and 0.05–0.1–0.5 mg/kg i.p respectively. Dexamethasone 0.5 mg/kg was used as reference compound. All data were expressed as mean ± SEM of n = 8/group. All statistical analyses were performed using one-way Anova followed by Dunnett's multiple comparison post test (*p<0.05, **p<0.01, ***p<0.001 vs saline-treated vehicle group).</p

Evaluation of CD3 infiltrates in ear sections.

<p>Immunostaining with CD3 antibody on ear (paraffin sections) of Balb/c females 8–12 weeks of age sensitized and challenged in a Oxazolone-induced CHS. In all panels, magnification 40× (scale bar: 20 µm). <b>A</b>) Vehicle group: the CD3 staining on ear section revealed an important T cells involvement. <b>B</b>) No reduction of T cell infiltrates in ear sections is observed for [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05 mg/kg. <b>C–D</b>) [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.5 and 1 mg/kg respectively were able to reduce T cell involvement to similar level as Dexamethasone. <b>E</b>) Dexamethasone 10 mg/kg was used as reference compound: clearly no staining is observed. <b>F</b>) Isotype control.</p

Evaluation of infiltrating inflammatory cells in ear sections.

<p>Haematoxylin and Eosin staining on paraffin ear sections of Balb/c females 8–12 weeks of age sensitized and challenged in a DNFB-induced CHS. 30′ after challenge, mice were treated with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05–0.5–1 mg/kg ip. Dexamethasone 0.5 mg/kg sc was used as reference compound. <b>4a</b>) Vehicle group: high level of infiltration, polymorphonuclear cells (PMN) mostly, dendritic cells (DCs) and Natural Killer cells (NK) as lowest populations. A detail of hyperplasia of epithelium, represented by an increase in keratinocyte layer thickness is also shown. <b>4b</b>) Cellular infiltrates and hyperplasia of epithelium are observed in ear sections of animals treated with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05 mg/kg. <b>4c</b>) [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.5 mg/kg were able to decrease cellular infiltrates, hyperplasia of epithelium (detail shown in the panel) and dermis size (edema) to similar level as Dexamethasone. <b>4d</b>) Reduction of cellular infiltrates and keratinocyte layers thickness is observed for [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 1 mg/kg. <b>4e</b>) Dexamethasone 0.5 mg/kg was used as reference compound. <b>4.1</b>) Control group: not-sensitized, normal skin conditions. In all panels, magnification 20× ( scale bar: 16.5 µm), details 40× (scale bar: 20 µm).</p

Expression of keratinocyte proliferation and differentiation markers in ear sections.

<p>Immunostaining for keratins was performed on paraffin sections of ear skin of Balb/c females 8–12 weeks of age sensitized and challenged by Oxazolone. Treatments with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 0.5 mg/kg via i.p. took place 30′ post challenge. Dexamethasone 10 mg/kg sc was used as reference compound. <b>A</b>) Vehicle: important hyperproliferation of keratinocyte layer: K6 is widely expressed and it is found supra-basal, K14 is constitutively expressed. K10 showed an inverse staining compared to K6. <b>B–C</b>) [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 0.5 mg/kg respectively were able to down-modulate K6 and K10 expression and to reduce hyperproliferation in a similar manner. K14 staining showed no changes. <b>D</b>) Dexamethasone 10 mg/kg used as reference compound: clearly no staining is observed. <b>E</b>) Isotype control. In all panels, magnification 20× (scale bar: 16.5 µm).</p

Therapeutic efficacy of [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 on ear swelling.

<p>Ear Swelling in head to head Oxazolone-induced CHS. CHS was induced by sensitization on shaved back with 3% Oxazolone at day 0. Challenge with 0.5% Oxazolone was done at day 5 to the right ear of sensitized mice. Mice were treated 30′ after irritation once with CCL5 antagonists at 0.5 mg/kg ip. Dexamethasone 10 mg/kg was used as reference compound. Results are given as mean ± SEM of n = 8 mice per group. All statistical analyses were performed using two-way ANOVA followed by Bonferroni's post test (***p<0.001 vs vehicle for each time point- 24,48,72 h).</p

Myeloperoxidase activity in ICD and CHS ear extracts.

<p>Determination of MPO was used as an indirect measure of neutrophils recruitment and content of ear tissue. Myeloperoxidase activity was tested respectively in ICD and CHS mouse models. In ICD mouse model, one biopsy of challenged ear (5 mm of diameter) of balb/c 8–12 weeks females was irritated with Croton oil and treated after irritation with <b>A</b>) [⁴⁴AANA⁴⁷]-CCL5 at 0.5–1–5 mg/kg and <b>B</b>) Met-CCL5 at 0.025–0.05–0.1–0.5 mg/kg via intraperitoneal injection. In DNFB-induced CHS (Fig. <b>C/D</b>) one biopsy of challenged ear (5 mm of diameter) of balb/c 8–12 weeks females was challenged by 0.2% DNFB and treated 30′ after irritation with [⁴⁴AANA⁴⁷]-CCL5 and Met-CCL5 at 0.05–0.5–0.1 mg/kg via intra-peritoneal injection. Results are given in % of vehicle for comparison of therapeutic efficacy of the two mutants, as mean ± SEM of n = 4 mice.</p