Study population
Eighteen patients with CPE from five veterinary hospitals in the metropolitan area of the Valley of Mexico were included between March and September 2019. All the puppies met the inclusion criteria of the research protocol (FTU/P3/19/03) as approved by the Ethics in Research Committee of Escuela Nacional de Ciencias Biológicas at Instituto Politécnico Nacional (ENCB-IPN), equivalent to IACUC regarding Handling and Care of Animals. All the tutors signed informed consent letters. The manuscript follows the recommendations in the ARRIVE guidelines and all experiments were performed in accordance with relevant guidelines and regulations.
The inclusion criteria of this study can be consulted in Table S1 of supplementary information. We included CPE patients under 6 months of age, regardless of sex and breed, that exhibited at least two clinical criteria of SIRS secondary to sepsis (hypothermia < 37.8 ºC, fever > 39.4 ºC, tachycardia > 140 bpm, tachypnea > 30 breaths/min, leukopenia < 5.5 x 109/L, and leukocytosis > 12.5 x 109/L) [7]. Patients were randomly assigned to any of the two study groups. The group with conventional treatment plus vehicle (CT+V, n = 7) or the conventional treatment plus immunomodulator group (CT+I, n = 11). Once the patients were approved for the study, they were admitted and remained under the care of veterinary doctors at the participant center.
Immunomodulator
The immunomodulator used is a hDLE, commercialized as Transferon (batch 18A03), produced for this protocol by Pharma-FT at ENCB-IPN (Mexico City, Mexico) [49]. Briefly, the leukocyte-platelet concentrates of healthy human donors were collected at blood banks in Mexico City and lysed through freezing/thawing cycles. After lysis, the active pharmaceutical ingredient (API) was obtained with tangential flow filtration, using a 10-kDa molecular weight cut-off cassette. It was especially formulated for this study with 0.5 mg/mL total protein concentration using UV absorbance at 280 nm. The final product passed the identity test by molecular size chromatography, endotoxin content by Limulus amebocyte lysate, and microbial limits according to the Pharmacopeia of the United Mexican States [50]. Then, the hDLE was stored in 10 mL sterile glass bottles with bromobutyl stoppers in laminar flow cabinets and handed over to the head researcher of the study along with the same number of vials containing water for injection (Laboratorios PiSa, Jalisco, Mexico). Both products were stored and transported in refrigeration (5 ± 3 ºC) to the administration site.
Administration of conventional treatment and immunomodulator
Patients were randomly assigned to two different groups once tutors gave their consent. Both groups were handled conventionally as recorded in the files of each patient. In addition, the control group (CT+V) received the vehicle, water for injection, and the other group (CT+I) was administered the immunomodulator (0.5 mg in 1 mL vehicle, daily). Treatments were administered subcutaneously in the interscapular region every 24 h for 5 days.
Sample collection: feces and whole blood
In all the cases, two fecal samples were collected using a sterile swab directly in the rectum. The first one was used as screening using rapid detection of CPV-2 by immunochromatography (Anigen Rapid CPV Ag Test kit, Bionote, South Korea) and the second helped confirm the diagnosis by endpoint polymerase chain reaction (PCR). The sample was placed in a test tube without anticoagulant (BD Vacutainer®, New Jersey, USA) with 1 mL sterile saline solution and kept at –20 ºC. It was then placed in a sterile microcentrifuge tube and centrifuged at 12,600 x g for 10 min. We added DNAzol (Invitrogen®, USA) to the supernatant obtained (400–600 µL) at a 1:1 ratio and kept it at –75 ºC until the extraction of genetic material.
Blood samples were extracted from the jugular vein at days 0, 1, 2, 3, 4, and 5. The blood was placed in tubes with anticoagulant EDTA (BD Vacutainer®, New Jersey, USA) and kept under refrigeration (5 ± 3 ºC) until its analysis. The amount obtained (1–3 mL) was adjusted to the patient’s weight per day, caring for the well-being of the animal. Once the hematological parameters were assessed, samples were centrifuged at 1,233 x g for 10 min. The plasma obtained was stored at –20 ºC until cortisol and neurotransmitters were quantified. The samples were obtained between 9 a.m and 12 p.m.
Confirmation of infection by CPV-2
Infection was confirmed at the Virology Laboratory of the Facultad de Medicina Veterinaria y Zootecnia (FMVZ) at Universidad Nacional Autónoma de México (UNAM). To extract the genetic material, 500 µL DNAzol (Invitrogen®, Massachusetts, USA) and 8 µL Proteinase K (Bioline, London, UK) were added at a concentration of 60 µg/mL to the stored sample (500 µL) at –75 ºC. The mixture was incubated at 56 ºC for 30 min and DNA was extracted according to the DNAzol reagent protocol, using 100 and 75% molecular biology grade ethanol (Sigma Aldrich, Darmstadt, Germany). The precipitate was hydrated with 30 µL DNAse-free water, and DNA was quantified by spectrophotometry in a NanoDrop spectrophotometer. The endpoint PCR was carried out following the protocol of Master Mix Platinum II PCR MM (Invitrogen®, Massachusetts, USA) in a MultiGene® Optimax thermal cycler with 30 denaturation cycles at 95 ºC for 30 s, annealing at 55 ºC for 30 s, and extension at 72 ºC for 30 s. The primers used, forward 5’-GAC CAG CTG AGG TTG GTT ATA G- 3’ and reverse 5’- GGT GCA TTT ACA TGA AGT CTT GG 3’, were directed to the gene coding for the capsid protein (VP2) of CPV-2 and created a 466-bp amplicon. They were designed by the research group at FMVZ, based on the publication by Balasubramaniam et al [51]. The electrophoresis was carried out in a 2% agarose gel stained with ethidium bromide for analysis in a UV transilluminator.
Clinimetric evaluation, weight, survival time, and days of hospitalization
The clinical evaluation took place across 5 days, between 8:00 a.m. and 11:00 a.m., according to the guidelines of the diagnostic methodology to perform a detailed clinical exam. The veterinarians in charge of the patients received training before the study to homologate clinical evaluation criteria. The parameters evaluated were temperature, mental health (alert and responsive, depressed or in coma), dehydration percentage, degree of abdominal pain, characteristics of feces (pasty stool, mucoid diarrhea, hemorrhagic diarrhea), presence and characteristics of vomit, and death 7. Each parameter was given a measuring scale; a higher score indicated the severity of the disease was greater. Table S2 of supplementary information specifies the clinical parameters evaluated as well as their scores. Weight was recorded during the clinical inspection.
Survival time record started 24 h after patients were admitted and ended at day 5. Days of hospitalization were quantified up to hospital discharge, which took place once the patients consumed feed and water in the absence of vomit for more than 24 h and showed no clinical signs of depression, fever, or hypothermia.
Hematological parameter assessment
Blood samples were sent to a private veterinary clinical laboratory for a complete hemogram. Hematocrit, hemoglobin, and total erythrocyte and leukocyte counts were assessed using a CELL-DYN® Emerald hematology analyzer (Abbot, Illinois, USA), which is based on electronic impedance and absorption spectrophotometry. The analyzer was calibrated and verified for the target species. The number of circulating neutrophils and lymphocytes was obtained from a blood smear with a differential leukocyte count. This information was used to obtain the absolute value based on the total number of leukocytes. Plasma proteins were identified in a conventional refractometer (RHC-200act, Mexico).
Plasmatic cortisol quantification
Plasmatic cortisol was determined in a private veterinary clinical laboratory. The plasma samples were processed in an Architect i2000SR immunoassay analyzer (Abbot, Illinois, USA). The method was based on a heterogeneous chemiluminescent microparticle immunoassay.
Quantification of plasmatic norepinephrine, epinephrine, and serotonin
Norepinephrine (NE), epinephrine (EP), and serotonin (SE) were determined at the Psychoimmunology Laboratory, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz. We extracted NE, EP, and SE from 250 µl plasma by adding 250 µl extraction buffer containing 5% ascorbic acid, 200 mM sodium phosphate, 2.5 mM L-cysteine, 2.5 mM EDTA, and 2.4 M perchloric acid. The mixture was incubated at –20 ºC for 10 min. The supernatants containing NE, EP, and SE were collected after centrifugation at 12,419 x g and 4 ºC for 10 min. They were processed by solid-phase extraction (SPE) using a Hypersep C18 cartridge (Thermo Scientific, Massachusetts, USA) without activation to retain lipids and recover neurotransmitters, and samples were passed through 0.22 µm filters. Neurotransmitter concentrations were identified by reversed-phase HPLC (RP-HPLC) in a system consisting of a PU-2089 plus pump (Jasco Inc., Japan) and an X-LC™ 3120FP fluorescence detector (Jasco Inc., Japan). The instruments were controlled using ChromNav software (Jasco Inc., Japan). Chromatographic runs were carried out with a Jupiter C18 column (300 Å, 5 µ, 4.6 x 250 mm, Phanomenex®) at 30 ºC. The column was in equilibrium with mobile phase A containing 0,1% trifluoroacetic acid in water. A linear gradient was run from min 5 to min 20 with mobile phase B containing 0,1% trifluoroacetic acid in acetonitrile. The flow rate was 0.8 mL/min. The fluorescence detector was set at a gain of 1000, attenuation of 32, response of 20 s, excitation at 280 nm and emission of 315 nm. The injection volume of the sample was 80–100 µL.
In silico analysis of the interaction between dog CXCR4 and human mUb and Ub∆GG
The crystallized structure of human CXCR4 contained in the Protein Data Bank (PDB; www.rcsb.org) ID. 4RWS was edited using Biovia Discovery Visualizer Studio software (BDVS, Discovery Studio Modeling Environment, 2017; Dassault Systèmes) to eliminate accessory proteins (vMIP2 and T4 lysozyme). In addition, the intramolecular loops of human CXCR4 were recovered using the Protein Preparation Wizard tool of Maestro v12.5 (Schrödinger Suite, Protein Preparation Wizard, 2021-1; Schrödinger). Edited human CXCR4 was used to model dog CXCR4 (Canis lupus familiaris; NP_00101041491.1) by homology in Modeller v9.25 [52]. The model with the lowest DOPE (Discrete Optimized Protein Energy) score was chosen among the five candidate models.
To carry out the homology modelling of mUb and Ub∆GG, the crystallized structure of 6KDU obtained from PDB was used as a model after edition with BDVS to eliminate accessory molecules (adenine 5-phosphoribose, Zn + 2, and Mg + 2). The model with the lowest DOPE score was chosen among the candidates for mUb and Ub∆GG.
To couple dog CXCR4 to human mUb or Ub∆GG, we used a masking template generated on PyMOL v2.0 (The PyMOL Molecular Graphics System version 2.0; Schrödinger) to direct the interaction specifically in the extracellular domain of CXCR4. The coupling was generated using the ClusPro 2.0 server [53], choosing the interaction mode Electrostatic-favored. The 10 major groups obtained were analyzed to identify the dissociation constant at 37 ºC with the PRODIGY server (prediction PROtein binding enerGY). The coupling visualization was produced with PyMOL software.
Statistical analysis
All statistical tests were run on GraphPad Prism v8.0.1 (San Diego, California, USA). In all cases, data were classified by group or evaluated time and were applied a Shapiro-Wilk test to assess normality. Once two groups with normal distribution were compared, one-way ANOVA and Tukey’s post-hoc test were applied. Alternatively, Kruskal-Wallis and Dunn’s post-hoc tests were used. To compare the age of patients and period of hospitalization, Student t and Mann-Whitney tests were applied, respectively.
When comparing weight in all the cases, we used the values resulting from the weight change percentage formula (see Equation 1)
(1)
Then, Kruskal-Wallis and Dunn’s post-hoc tests were carried out. The data analysis of the clinical score was using a Kruskal-Wallis test was done. The survival was assessed with a Kaplan-Meier survival curve and a Gehan-Breslow-Wilcoxon post-hoc test. To evaluate the differences induced by the treatments evaluated in circulating neutrophil and lymphocyte counts, we used the values obtained on days 0, 2, and 4. The neurotransmitter comparison was evaluated on days 0, 1, and 2 through a Kruskal-Wallis and Dunn’s post-hoc tests. In all cases, there was a statistical difference when P ≤ 0.05, and data were described with mean ± SD.