Mitigation of septic shock in mice and rhesus monkeys by human chorionic gonadotrophin-related oligopeptides

Authors


B. A. ‘t Hart, Department of Immunobiology, BPRC, Lange Kleiweg 139, 2288 GJ Rijswijk, the Netherlands.
E-mail: hart@bprc.nl

Summary

The marked improvement of several immune-mediated inflammatory diseases during pregnancy has drawn attention to pregnancy hormones as potential therapeutics for such disorders. Low molecular weight fractions derived from the pregnancy hormone human chorionic gonadotrophin (hCG) have remarkable potent immunosuppressive effects in mouse models of diabetes and septic shock. Based on these data we have designed a set of oligopeptides related to the primary structure of hCG and tested these in models of septic shock in mice and rhesus monkeys. We demonstrate that mice exposed to lipopolysaccharide (LPS) and treated subsequently with selected tri-, tetra-, penta- and hepta-meric oligopeptides (i.e. MTR, VVC, MTRV, LQGV, AQGV, VLPALP, VLPALPQ) are protected against fatal LPS-induced septic shock. Moreover, administration of a cocktail of three selected oligopeptides (LQGV, AQGV and VLPALP) improved the pathological features markedly and nearly improved haemodynamic parameters associated with intravenous Escherichia coli-induced septic shock in rhesus monkeys. These data indicate that the designed hCG-related oligopeptides may present a potential treatment for the initial hyperdynamic phase of septic shock in humans.

Introduction

In immunological terms, pregnancy is an improbable symbiosis of two major histocompatibility complex (MHC)-incompatible individuals. Pregnant women are eminently capable of combating infections and often produce antibodies against paternal alloantigens of the fetus, demonstrating that they are fully immunocompetent. Nevertheless, a remarkable improvement of several immune-mediated inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis, is often observed during pregnancy [1,2]. These well-known, albeit poorly understood, features are suggestive for a selective modulation of the immune system in such a way that harmful immune processes to mother and fetus are suppressed, while beneficial immune processes are not affected. Certain hormones that are produced exclusively during pregnancy, such as oestriol or human chorionic gonadotrophin (hCG), have been held responsible for the selective modulation of adverse immunological functions [2–4].

Human chorionic gonadotrophin (hCG) is a heterodimeric glycoprotein produced by trophoblasts [5]. Its main hormonal function is to preserve the lining of the uterus, which is needed for the healthy development of the implanted embryo [5]. Activity-guided purification of commercial hCG preparations has shown that oligomeric degradation products represent a significant part of the immunosuppressive activity in immune-mediated inflammatory disease models [6,7], but they lack the endocrine effects of the complete protein. These capacities make hCG-derived oligopeptides a potentially interesting source of therapeutics for immune-mediated inflammatory diseases.

The aim of the current study was to test whether such oligopeptides related to hCG can also reduce the severity of lipopolysaccharide (LPS)-induced and Escherichia coli-induced septic shock. Contamination of the bloodstream with bacteria or bacterial endotoxins (e.g. LPS) can cause massive activation of complement and the release of proinflammatory cytokines, such as tumour necrosis factor (TNF)-α[8]. Irrespective of the origin (infectious or non-infectious), this leads to life-threatening haemodynamic changes, such as tachycardia and hypotension culminating in multi-organ failure. Without adequate treatment, septic shock can be lethal within a few days after onset.

Sepsis is the second leading cause of death in intensive care units (ICUs) in the United States [9,10] and is responsible for about 10% of overall deaths annually [11]. There is a high unmet need for a safe and effective treatment of septic shock, particularly to prevent the irreversible multiple organ failure stage.

For the present study we designed a set of hCG-related oligopeptides all related to loop 2 of the β-chain, namely MTR, MTRV, LQG, LQGV, VLPALP, VLPALPQ and VVC, as well as an alanine variant of LQGV, namely AQGV. This selection was based on known nick sites in this loop [12–14]. The oligopeptides were first tested in a murine LPS infusion model for attenuation of septic shock. The mouse study yielded three peptides that rescued 100% of the mice from death after 48 h, namely AQGV, LQGV and VLPALP. These three oligopeptides were tested subsequently as a cocktail for the rescue of rhesus monkeys from septic shock induced by infusion of living E. coli. Administration of a single dose of the oligopeptide cocktail delayed the manifestation of septic shock symptoms, reduced pathomorphological changes markedly in the gastrointestinal tract and nearly normalized the haemodynamic parameters.

Collectively, the reported data show that the tested oligopeptides are well tolerated and display promising therapeutic effects in two relevant animal models of septic shock.

Materials and methods

Animals

Mice.  Female 8–16-week-old BALB/c mice were used. The mice were bred under specific pathogen-free conditions according to the protocols described in the Report of the Federation of European Laboratory Animal Science Association's (FELASA) working group on animal health [15]. The mice were bred and used in the facilities for experimental animals of the Erasmus MC. The mice had access to pelleted food (Hope Farms, Woerden, the Netherlands) and sterilized water ad libitum.

Rhesus monkeys.  Eight healthy female rhesus monkeys (Macaca mulatta) were purchased from the purpose-bred breeding colony at the Biomedical Primate Research Centre. Individual data of the monkeys are given in Table 1. Prior to inclusion in the experiment, monkeys received a physical health check and were tested for haematological, serological and microbiological abnormalities. Only monkeys that were declared healthy by the institute's veterinarian staff were entered into the experiment.

Table 1.  Individual rhesus monkey data and treatment regimen.
Treatment groupAnimal IDGenderDate of birthDate experimentAge (years)Weight (kg)
I Control; termination after 8 h (mean age 7·1 years)Ri429F15 January 199628 November 20015·85·6
Ri274F24 February 199824 August 20057·54·9
Ri8012F1 August 199820 September 20068·15·8
II Oligopeptide cocktail; termination after 8 h (mean age 7·1 years)Ri459F21 June 199628 November 20015·45·4
Ri7046F3 July 199820 September 20068·25·7
Ri11152F7 November 199827 September 20067·85·3
III Oligopeptide cocktail; recovery allowed (mean age 8·0 years)Ri 427F17 June 199628 November 20015·44·8
C 152F14 February 199524 August 200510·56·2

Oligopeptides

Selection was based on either the known preferential cleavage sites or known nick sites of the sequence MTRVLQGVLPALPQVVC (aa41–57) of loop 2 (Fig. 1) of the β-subunit of hCG [12–14,17]. Oligopeptides selected for the in vivo studies reported in this publication were the most effective in pilot studies, i.e. MTR (aa41–43), MTRV (aa41–44), LQG (aa45–47), LQGV (aa45–48), AQGV (alanine replaced oligopeptide of LQGV; aa45–48), VLPALP (aa48–53), VLPALPQ (aa48–54) and VVC (aa55–57). Oligopeptides were synthesized (Ansynth BV, Roosendaal, the Netherlands) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology with a 2-chlorotritylchloride resin as the solid support.

Figure 1.

Structure of β-human chorionic gonadotrophin (hCG) with loop 2 and the amino acid sequence of loop 2 indicated. Adapted from Lapthorn et al. [16]. Arrows point to the preferential cleavage sites in loop 2 (aa41–57).

LPS-induced acute septic shock in mice

Female 8–12-week-old BALB/c mice (n = 5–6 per group) were injected intraperitoneally (i.p.) with a lethal dose of LPS (8 mg/kg; E. coli 026:B6; Difco Laboratories, Detroit, MI, USA). This procedure leads to 100% death within 84 h. Mice were treated subsequently with either a single i.p. dose of phosphate-buffered saline (PBS) or oligopeptide (5 mg/kg) in PBS at 2 h or 24 h post-LPS challenge. The optimal oligopeptide dose for this model has been defined in a previous study [7]. Survival of the mice was monitored for 84 h at least three times per day. Mice were scored in a semi-quantitative fashion (scores 1–6) for sickness severity, as described previously [7].

LPS and CD3-induced proliferation of splenocytes

Sixteen-week-old female BALB/c mice (n = 5 per group) were injected i.p. with either LQGV or AQGV (5 mg/kg) or the equivalent volume of solvent (PBS). After 1 h of treatment the mice were killed and spleens were isolated. Five spleens from a group were pooled and splenocytes were isolated. For T cell stimulation splenocytes were cultured (2·5 × 105 cells/well) in 96-well flat-bottomed plates (0·2 ml) in triplicate and stimulated with anti-CD3 (145-2C11, 10 mg/ml) in combination with interleukin (IL)-2 (Biosource, CA, USA; 40 U/ml). For B cell and macrophage stimulation, splenocytes were cultured (5 × 105 cells/well) in 96-well round-bottomed plates (0·2 ml) in triplicate and stimulated with 10 mg/ml of LPS (E. coli 026:B6; Sigma; 69H4022) or with PBS. Splenocytes were then incubated at 37°C in 5% CO2 for 12, 24, 36 or 48 h. During the final 8–12 h of culture 0·5 mCi of [3H]-thymidine ([3H]TdR) per well was added and the incorporation of [3H]TdR was measured on a beta-plate counter.

E. coli-induced acute septic shock in adult rhesus monkeys

Bacteria.  The E. coli strain used for septic shock induction was purchased from the American Type Culture Collection (ATCC) (E. coli 086a: K61 serotype; ATCC 33985). In a control experiment the strain had proved equally sensitive to complement-mediated lysis in fresh human and rhesus monkey serum (data not shown).

Prior to the experiment a fresh E. coli culture was set up in brain heart infusion (BHI) culture broth. The E. coli strain was cultured for 1 day, harvested and washed five times to remove free endotoxin. Just prior to infusion into the monkey a sample of the bacteria suspension was collected to assess the concentration and viability. To this end, serial dilutions of the E. coli stock were plated on BHI agar and cultured overnight at 37°C. The colonies on each plate were counted and the numbers of colony-forming units (CFU)/ml were calculated. The body weight measurement on the day of the experiment was used to calculate the E. coli dose (1010 CFU/kg). The E. coli stock was suspended in pyrogen-free isotonic saline (NPBI, Emmer Compascuum, the Netherlands) adjusted to the required concentration, and kept on ice until infusion. Just prior to infusion the total dose was resuspended in a volume of 50 ml isotonic saline of room temperature.

Preparation of the animals.  Monkeys were fasted overnight prior to the experiment. On the morning of the experiment the monkeys were sedated with ketamine hydrochloride (AST Pharma, Oudewater, the Netherlands) and transported to the operation chamber. Monkeys were placed on their side on a temperature-controlled heating pad to support body temperature. Body core temperature was monitored using Ohmeda Excel 210 SE anaesthesia equipment (Datex-Ohmeda, Hoevelaken, the Netherlands). Monkeys were intubated orally and allowed to breathe freely. The femoral or cephalic vein was cannulated and used for infusion of isotonic saline, live E. coli, antibiotics and the oligopeptide cocktail. Fluid loss was compensated by infusing isotonic saline containing 2·5% glucose (Fresenius, 's-Hertogenbosch, the Netherlands) at a rate of 3·3 ml/kg/h. To avoid discomfort to the monkeys they were kept anaesthetized using O2/N2O/isoflurane inhalation anaesthesia during the two E. coli infusions and a 6-h observation period following E. coli challenge. The monkeys were kept under constant veterinary inspection during the complete time-course of the experiment.

Induction of septic shock.  A 1-h time-period was included to monitor baseline values of heart rate and blood pressure prior to infusion of E. coli. Septic shock was induced by infusion of a fatal dose (1010 CFU per kg body weight) of live E. coli over a period of 2 h followed immediately by intravenous (i.v.) administration of the bacteriostatic agent Enrofloxacin [dose: 9 mg/kg; Enrofloxacine (Baytril 2·5%); Bayer, Mannheim, Germany] to kill surviving bacteria and to synchronize shock induction. In two independent (dose titration) studies with the chosen E. coli strain, a dose of 1010 CFU/kg body weight-induced acute shock leading to death about 8 h after start of the infusion (unpublished observations). Thirty minutes after the start of E. coli infusion the monkeys received a single i.v. bolus injection (1 ml per kg body weight) of oligopeptide cocktail containing LQGV, AQGV and VLPALP at a concentration of 5 mg/ml each; the dose was extrapolated from initial studies in mice [7]. The peptide solution was injected into the i.v. cannula via which isotonic saline was infused continuously. For experimental reasons explained in the Results section, monkey Ri427 was allowed to recover after 8 h and received no further treatment. Monkey C152 was allowed to recover after 8 h and received a second treatment at this time-point and a third treatment at 24 h.

Pathology

Post-mortem examination of all monkeys was conducted immediately after the monkeys were killed. The monkeys underwent gross necropsy in which the abdominal and thoracic cavity was opened and internal organs were examined in situ. Pictures were made of the internal organs of some of the monkeys. Tissues of all organs were preserved in neutral aqueous 4% solution of phosphate-buffered formaldehyde and processed subsequently for histopathological examination by a pathologist.

Cytokine production

Cytokine levels in plasma were analysed using Cytometric Bead Array™ (CBA; BD Biosciences, San Diego, CA, USA). TNF-α, IL-1β, IL-6 and IL-8 were detected using the human inflammation CBA kit. Tests were performed according to the manufacturer's instructions and known to have cross-reactivity with rhesus monkey. The results were expressed as pg/ml. The limits of detection were as follows: 2·6 pg/ml TNF-α, 7·2 pg/ml IL-1β, 2·5 pg/ml IL-6, 3·6 pg/ml IL-8.

Statistics

Data were analysed by two-tailed Fisher's exact test and unpaired Student's t-test. Data differences were considered significant at P < 0·05.

Ethics

The protocols of the mouse studies have been reviewed and approved by the Animal Research Ethics Committee of Erasmus MC (Rotterdam, the Netherlands). The protocols of the rhesus monkey studies have been reviewed and approved by the Animal Research Ethics Committee of the Biomedical Primate Research Centre (Rijswijk, the Netherlands).

Results

Effects of hCG-related oligopeptides on LPS-induced mortality in mice

Intraperitoneal injection of LPS in BALB/c mice induced a fatal septic shock leading to 100% mortality within 84 h. Several synthetic oligopeptides related to loop 2 of the hCG β-chain (Fig. 1) had a marked protective effect on the morbidity and mortality induced by the LPS administration. In the first series of experiments a single dose (5 mg/kg body weight) of the individual oligopeptide or PBS as control was administered 2 h after the LPS injection. At the 48 h time-point all (17 of 17) LPS-injected mice receiving PBS were dead (Fig. 2a, PBS). Oligopeptides LQGV and the alanine-substituted derivative AQGV were equally effective as VLPALP in rescuing mice (17 of 17) from LPS-induced septic shock (Fig. 2a). In a previous study [7] the latter peptide was found to prolong survival time beyond 72 h when administered 2 h after a lethal challenge of LPS. In the second series of experiments a single dose of the individual peptides was given 24 h after the LPS administration. Here, five of the eight oligopeptides prevented mortality completely (Fig. 2b). Of all eight oligopeptides tested in this model, only AQGV rescued all (17 of 17) mice from septic shock when administered early (at 2 h) as well as late (at 24 h) after LPS-injection.

Figure 2.

Survival of mice with lipopolysaccharide (LPS)-induced septic shock after treatment with a single dose of different human chorionic gonadotrophin (hCG)-related oligopeptides. Mice were treated with the following oligopeptides (5 mg/kg body weight i.p.): phosphate-buffered saline (PBS) (red triangle) MTR (aa41–43; black circle, dashed line), MTRV (aa41-44; brown circle, dashed line), LQG (aa45–47; green triangle), LQGV (aa45–48; blue diamond), AQGV (alanine replaced oligopeptide of LQGV; aa45–48; orange diamond), VLPALP (aa48–53; black diamond), VLPALPQ (aa48–54; black triangle) and VVC (aa55–57; green circle, dashed line). Mice were treated either 2 h (a) or 24 h (b) after administration of LPS intraperitoneally. Cumulative data are presented from three independent experiments (five to six animals per group/experiment). The survival percentages at 84 h were highly significant (P = 0·000001, two-tailed Fisher's exact test) for peptides LQGV, AQGV and VLPALP at 2 h and for peptides MTR, MTRV, VLPALPQ, VVC and AQGV compared to the PBS group.

Signs of sickness were apparent in all LPS-treated mice, but the course and severity differed between individual animals. In order to quantify the observed differences, a semiquantitative sickness scoring system was used, with severity scores ranging from 0 to 6. In these experiments, mice injected i.p. with 8 mg LPS/kg body weight reached a sickness score of 2 or 3 within 14 h. At 24 h, most of the mice had reached a sickness score above 4. Thereafter the mice progressed gradually to a sickness score of 5 or had succumbed (score 6). Injection of LQGV, VLPALP or AQGV 2 h after LPS injection reduced the average sickness scores to less than 2 at 84 h after LPS injection (Table 2). The other oligopeptides were less effective. Although LQGV, VLPALP and AQGV all rescued mice from the fatal outcome of septic shock and reduced sickness scores, AQGV treated mice had significantly (P < 0·01) lower sickness scores at 48 h after LPS injection than LQGV- and VLPALP-treated mice. Treatment of BALB/c mice with either MTR, MTRV, VLPALPQ, VVC or AQGV at 24 h after LPS injection rescued all mice from septic shock. Here, mice treated with AQGV also had significantly (P < 0·01) lower sickness scores 48 h after LPS injection compared to mice treated with MTR, MTRV, VLPALPQ and VVC. Remarkably, AQGV not only prevented mortality but also reduced the average sickness score from 4·2 at the time of treatment to 1·5 60 h later. Two days later all mice that were rescued from septic shock death by the oligopeptide treatment had recovered and had a sickness score of 1 or no longer had any signs of sickness. The other oligopeptides were less effective in reducing sickness scores (MTR, MTRV, VLPALPQ, VVC) and preventing mortality (LQG, LQGV, VLPALP).

Table 2.  Average sickness scores of mice with lipopolysaccharide (LPS)-induced septic shock after treatment with a single dose of different human chorionic gonadotropin (hCG)-related oligopeptides.
TreatmentAverage sickness scores after time interval (h)*
2 h after LPS induction24 h after LPS induction
014244884014244884
  • *

    Mice were treated with the various oligopeptides [5 mg/kg body weight intraperitoneally (i.p.)] 2 h or 24 h after administration of LPS i.p. (n = 6).

  • Indicates that none the mice had survived at that time-point.

  • Indicates that the sickness scores are significantly higher than the AQGV-treated group (P < 0·01). Mice were scored for sickness severity as described previously (7) using the following criteria: score 1: percolated fur, but no detectable behaviour differences compared with untreated control mice; score 2: percolated fur, huddle reflex, responding to stimuli (such as tap on cage) and just as active during handling as untreated control mice; score 3: slower response to tap on cage, and passive or docile behaviour when handled, but still curious when alone in a new setting; score 4: lack of curiosity, little or no response to stimuli, and defect mobility; score 5: laboured breathing and impaired righting reflex; score 6 was defined as death. The results illustrated are from a single experiment and representative of at least three independent sets of experiments (each group: n = 6). PBS: phosphate-buffered saline.

PBS0·02·84·30·03·04·5
MTR0·02·53·04·22·30·02·84·33·83·0
MTRV0·02·74·04·03·50·03·04·83·33·2
LQG0·03·04·70·02·74·7
LQGV0·02·53·02·71·20·02·84·2
VLPALP0·02·53·22·81·70·02·54·0
VLPALPQ0·03·24·50·03·24·52·72·7
VVC0·02·84·24·43·70·03·34·72·72·8
AQGV0·02·02·31·71·00·02·74·21·81·5

LPS and CD3-induced proliferation of splenocytes

The effect of LQGV and AQGV on systemic immune activation was assessed by measuring splenocyte proliferation in response to a T cell stimulator (anti-CD3 and IL-2) and a B cell and macrophage stimulator (LPS).

Splenocytes derived from LQGV and AQGV-treated mice had a significantly (P < 0·05) reduced in vitro proliferative response to CD3/IL-2 after 24 h of stimulation (Fig. 3). Splenocytes derived from AQGV-treated mice also showed a significantly (P < 0·05) reduced in vitro proliferative response to LPS (Fig. 3). Both LQGV and AQGV treatment were associated with a significantly (P < 0·05) reduced basal proliferation level (data not shown).

Figure 3.

The effect of in vivo treatment of BALB/c mice with the oligopeptides LQGV and AQGV (5 ml/kg) on the in vitro CD3/interleukin (IL)-2 (a) and (b) lipopolysaccharide (LPS)-induced splenocyte proliferation. Both oligopeptides significantly (*P < 0·05) reduced in vitro the capacity of the splenocytes to proliferate upon CD3/IL-2 stimulation. However, splenocytes derived from AQGV peptide-treated mice also showed significantly (*P < 0·05) reduced in vitro proliferative response to LPS after 24 h of stimulation, while no significant differences in in vitro proliferative response of splenocoytes derived from LQGV peptide-treated mice to LPS were found. The results presented are from a single experiment and representative of at least three independent sets of experiments (n = 5).

Treatment of E. coli-induced septic shock in rhesus monkeys

The three most promising oligopeptide candidates from the septic shock experiments in mice – AQGV, LQGV and VLPALP – were selected for evaluation in a preclinical model of E. coli-induced septic shock in rhesus monkeys.

Control animals (group I, consisting of monkeys numbered Ri429, Ri274, Ri8012) received 0·9% sodium chloride solution starting at 30 min after initiation of the infusion of the E. coli. These monkeys were not allowed to recover from anaesthesia and were killed 8 h after start of the infusion. Three other monkeys (group II, consisting of monkeys numbered Ri459, Ri7046, Ri11152) received a bolus injection of a cocktail containing the three selected oligopeptides (LQGV, AQGV, VLPALP; 5 mg/kg for each of the oligopeptides) dissolved in 0·9% sodium chloride solution 30 min after initiation of the E. coli infusion. Throughout the experiment the clinical condition of the monkeys was monitored. At 6 h the veterinarian responsible was asked whether the clinical condition would allow recovery from anaesthesia. Although this was approved in two of the three treated monkeys, these were nevertheless killed to compare the organ histopathology with the control monkeys. Two additional monkeys (group III, numbered Ri427, C152), which also received a bolus injection of the oligopeptide mix 30 min after initiation of the E. coli infusion, were allowed to recover from the anaesthesia at the veterinarian's consent on the basis of the clinical condition, and remained under constant supervision.

The clinical observations made by the veterinarian are summarized in Table 3. In the saline-treated control monkeys (group I) the septic shock crisis started 30 min after start of the E. coli infusion (= time-point ‘post-E. coli’). The condition of the animals worsened rapidly and displayed the typical signs of a septic shock crisis, characterized by highly unstable blood pressure and oxygen saturation (data not shown), disturbed electrocardiogram (ECG) output and several other abnormal clinical parameters (Table 3). The condition of the monkeys treated with the oligopeptide cocktail (groups II and III) was much more stable, except in monkey Ri11152. During the first 8 h of the experiment, four of five peptide-treated monkeys had a stable heart rate (data not shown), normal ECG and normal or depressed respiration (Table 3); however, depressed respiration was deep but regular. The outlier peptide-treated monkey Ri11152 displayed signs of a transient crisis which was far less serious than in the saline-treated control monkeys (Table 3). This monkey was deemed unfit for recovery from anaesthesia and was killed at 8 h.

Table 3.  Clinical observations during the first 8 h after induction of septic shock in rhesus monkeys.
TreatmentAnimal IDClinical observationVeterinarian
  1. ECG: electrocardiogram; PBS: phosphate-buffered saline.

Group I
PBS; termination after 8 h
Ri429At 8 h the animal showed faecal vomiting and convulsions. No pulse and arrhythmia. An abnormal ECG. During the last hours the animal displayed forced respiration and decreased blood clotting as indicated by continued bleeding after blood extractionNo recovery allowed
Ri274A progressively decreasing heart rate and blood pressure was observed. The animal also developed arrhythmia. The oxygen saturation could hardly be measured and at the end decreased rapidly. The animal became increasingly unstableNo recovery allowed
Ri8012After administration of the Esherichia coli there was a rapid development of oedema above the eyes and also in the lungs. In the final phase the animal displayed forced respiration and bronchi at auscultation indicative of oedema of the lungs. Abnormal ECG. The animal became increasingly unstableNo recovery allowed
Group II
Peptide cocktail; termination after 8 h
Ri459The animal had a good pulse and a normal ECG. The heart sounded normal and lungs sounded clean. Although the animal displayed depressed respiration, the respiration was deep and regularRecovery allowed
Ri7046The animal remained stable during the observation period. The animal developed mild oedema after infusion of Escherichia coli. The animal showed mild depressed respiration, but it was deep and regularRecovery allowed
Ri11152Initial stable clinical parameters became less stable after infusion of Escherichia coli. After administration of Enrofloxacin a stable increased heart frequency was measured but blood pressure became difficult to assess. In the later phase of the disease oedema was diagnosed after auscultation, necessitating repositioning of the animal on his side so that the animal could breath more freely. In the final phase the lungs sounded normal but an abnormal ECG was measuredNo recovery allowed
Group III
Peptide cocktail; recovery allowed
Ri 427The animal had a good pulse and a normal ECG. The heart sounded normal. The left lung displayed a slight murmur but overall sounded good. This animal had a normal regular respirationActually recovered
C 152The animal had a good pulse and a normal ECG. The heart sounded normal and the lungs sounded clean at 8 h. This animal had a normal respiration and stable clinical parametersActually recovered

Monkey Ri7046 showed only mild signs of septic shock, but although deemed fit for recovery it was killed at 8 h post-E. coli infusion. Monkey Ri459 was in good clinical condition 8 h post-E. coli infusion, but was nevertheless killed to allow pathological examination and comparison with control animals. The monkeys in group III (Ri427 and C152) were allowed to recover from anaesthesia and remained free of septic shock symptoms for 33 h and 36 h, respectively, and succumbed with serological evidence of organ failure. From this group, Ri427 had not received additional oligopeptide treatment while C152 had received two additional injections at 8 h and 24 h post-E. coli infusion.

Taken together, three of three saline treated-monkeys developed severe septic shock within 6 h after E. coli infusion. In contrast, three of five monkeys treated with the peptide cocktail remained completely devoid of septic shock symptoms during the initial observation period of 8 h. The two monkeys that were allowed to recover from anaesthesia (Ri427 and C152) finally developed signs of multiple organ failure. In the one oligopeptide cocktail-treated monkey that showed clinical signs of septic shock, these were far less serious than in the saline-treated control monkeys.

Effects of hCG-related oligopeptide treatment on gross pathology and histology of vital organs

Lungs.  Gross examination at necropsy of the saline-treated monkeys (Ri274, Ri429 and Ri8012) revealed dark red lungs. Microscopically prominent pulmonary oedema, vascular congestion and multi-focal areas of extravasated red blood cells (haemorrhages) were observed (see Fig. 4a). In the oligopeptide-treated monkey (Ri11152) that developed septic shock the lung had a similar haemorrhagic appearance. In the other two oligopeptide-treated animals (Ri459, Fig. 4b; and Ri7046, which had mild septic shock-related clinical symptoms) that were euthanized the macro- and microscopic changes were reduced markedly and moderately, respectively.

Figure 4.

Reduced lung pathology treatment with oligopeptide cocktail. The picture shows control monkey Ri 429 (a) and the oligopeptide-treated monkey Ri 459 (a). The marked difference in pulmonary oedema, vascular congestion and haemorrhages is easily visible.

Gastrointestinal tract.  Macroscopically, the wall of the small and large intestine of saline-treated monkeys Ri274 and Ri8012 (Fig. 5A) was moderately thickened (oedematous) with multi-focal to coalescing bright red areas present predominantly on the intestinal mucosa accompanied by scant greenish liquid faecal material in the lumen. Microscopically, the intestinal mucosa of these monkeys exhibited moderate vascular congestion, multi-focal areas of haemorrhage and mild to moderate oedema with mildly increased numbers of multi-focal lymphoplasmacytic infiltrates (Fig. 6C). Compared to the saline-treated monkeys, the intestinal mucosa of the oligopeptide-treated monkeys Ri459, Ri7046 and Ri11152 showed similar but milder gross lesions (Fig. 5C,E). Microscopically, monkey Ri11152 (Fig. 6D) exhibited only mild vascular congestion and oedema with few small haemorrhagic foci.

Figure 5.

Diminished septic shock-related gross pathological findings after treatment of rhesus monkeys with a cocktail of three human chorionic gonadotrophin (hCG)-related oligopeptides. Escherichia coli-related septic shock was induced in eight rhesus monkeys. Six animals were killed at 8 h in order to evaluate the effect of treatment with hCG-related oligopeptides at the level of histopathology after the acute phase of septic shock relative to phosphate-buffered saline (PBS)-treated control monkeys. A significant difference was observed between tissues from control monkeys (e.g. Ri8012; A, B) and treated monkeys (e.g. Ri7046: C, D; Ri11152: E, F). The gastric mucosa of control monkey Ri8012 (B) exhibits marked oedema, congestion and haemorrhage of the cardia (c) and fundus (f) and multiple petechial haemorrhages in the pylorus (p), while the treated monkeys (e.g. Ri7046; D and Ri11152; E) have milder lesions in the cardia and pylorus. Furthermore, the intestinal wall of the large intestine of control animals (e.g. Ri8012; A) exhibits multi-focal to coalescing areas of haemorrhage and oedema, while treated animal Ri7046 (C) shows milder lesions and treated animal Ri11152 (F) exhibits only scattered petechial haemorrhagic foci.

Figure 6.

Decreased severity of septic shock-related microscopic lesions after treatment of rhesus monkeys with a cocktail of three human chorionic gonadotropin (hCG)-related oligopeptides. Microscopic examination of tissues of control monkey Ri8012 (A, C, E) demonstrates marked vascular hyperaemia, vasodilation, multi-focal haemorrhages, marked oedema, mildly increased number of multi-focal lymphoplasmacytic infiltrates and sloughing (necrosis and loss) of epithelium in the lamina propria of the gastric mucosa (A) while the treated monkey Ri11152 (B) displays significantly minimized changes. The large intestinal mucosa in control monkey Ri8012 (C) exhibits moderate vascular dilation, active mucosal congestion, multi-focal areas of extravasated red blood cells (haemorrhage), mild to moderate oedema and mildly increased lymphoplasmacytic infiltrates, while the treated monkey Ri11152 (D) exhibits mild vascular hyperaemia and oedema with few small haemorrhagic foci. The hepatic parenchyma of control monkey Ri8012 (E) shows variable number of multi-focal sinusoidal neutrophils (periportal, random sinusoidal and intravascular), multi-focal hyperaemic blood vessels, multi-focal haemorrhage and oedema, while microscopic hepatic alterations of the treated monkey Ri11152 (F) are restricted to the presence of occasional sinusoidal neutrophils.

In the stomach of the saline-treated monkeys Ri429, Ri274 and Ri8012 (Fig. 5B) we observed severely affected gastric mucosa, exhibiting marked oedema, congestion and haemorrhage of the cardia and fundus and multiple petechial haemorrhages in the pylorus. Microscopically, these alterations were consistent with marked vascular congestion of the gastric mucosa and submucosa, vasodilatation in the lamina propria of the gastric mucosa (Fig. 6A), multi-focal haemorrhages, marked oedema and occasional sloughing (necrosis and loss) of gastric epithelium (degeneration and loss of few parietal and chief cells), accompanied by multi-focal lymphoplasmacytic infiltrates.

The oligopeptide-treated monkeys Ri7046 and Ri11152 showed only minimal gross lesions. Monkey Ri7046, compared to the control monkey Ri8012, showed milder lesions: mild congestion and few small haemorrhagic foci in the cardia and pylorus (Fig. 5D). Monkey Ri11152 exhibited only minimal to mild vascular congestion and oedema with few petechial haemorrhagic foci (Fig. 5F). In the cardia of monkey Ri274 (data not shown) only few petechial haemorrhages were found. Microscopically, these gross lesions were comprised of scattered congested blood vessels, few foci of extravasated red blood cells and mild oedema.

Liver.  The liver of the oligopeptide-treated and control monkeys was unremarkable macroscopically. Microscopic abnormalities in the hepatic parenchyma of saline-treated monkeys Ri429, Ri274 and Ri8012 (Fig. 6E) consisted of moderate to marked sinusoidal and intravascular neutrophilic granulocytosis, multi-focal congested vessels and multi-focal pericentral haemorrhages. The only observed microscopic alterations in the liver of oligopeptide-treated monkeys Ri459, Ri7046 and Ri11152 (Fig. 6F) were occasional sinusoidal neutrophils and few apoptotic hepatocytes.

Other organs.  The myocardium of the control monkeys Ri429, Ri274 and Ri8012 exhibited multi-focal degeneration of myocardiocytes and focal haemorrhage in Ri274. On the other hand, no significant abnormalities were present in the myocardium of the oligopeptide-treated monkeys Ri459, Ri7046 and Ri11152 (data not shown). Similarly, the pancreas of the control-treated monkeys Ri429, Ri274 and Ri8012 exhibited mild interlobular oedema and random degeneration of acinar cells, while no significant findings were present in the pancreas of the oligopeptide treated monkeys Ri459, Ri7046 and Ri11152 (data not shown).

Overall, the most serious pathomorphological changes related to septic shock were observed in the saline-treated control monkey Ri8012. Similar but less severe changes were observed in the two other monkeys from the control group. The least severe abnormalities were observed in the oligopeptide-treated monkeys Ri459, Ri7046 and Ri11152. These findings indicate a marked beneficial effect of the AQGV–LQGV–VLPALP oligopeptide cocktail on the course of this experimentally induced septic shock.

Effect of hCG-related oligopeptide treatment on the cytokine burst in rhesus monkeys

The effect of the AQGV–LQGV–VLPALP oligopeptide treatment was also evaluated on the plasma levels of proinflammatory cytokines of the control and oligopeptide-treated monkeys. E. coli infusion was associated with an increase of IL-1β, TNF-α, IL-6 and IL-8 plasma levels over time in both control and oligopeptide-treated monkeys. The plasma levels of these proinflammatory cytokines did not differ markedly between the oligopeptide group and the untreated monkeys (Fig. 7).

Figure 7.

Plasma levels of inflammatory cytokines. Escherichia coli infusion of rhesus monkeys was associated with an increase of interleukin (IL)-1β, tumour necrosis factor-α, IL-6 and IL-8. The plasma levels of these proinflammatory cytokines did not differ markedly between the oligopeptide-treated monkeys and the untreated monkeys.

Discussion

In previous studies the immunomodulatory activity of the pregnancy hormone hCG in LPS-induced acute septic shock and in diabetes in non-obese diabetic (NOD) mice was mapped to a 400–2000 Dalton fraction in pregnancy urine [6,7]. It was hypothesized that the active fraction contained oligopeptides derived from the sequence MTRVLQGVLPALPQVVC (residues 41–57) of loop 2 of the hCG-β chain [17]. ‘Missing’ of loop 2 from the β-subunit (as in hCG β-core fragment) and ‘nicking’ of the β-subunit in the loop 2 region of the molecule, specifically between residues 44–49, can both reduce the biopotency of hCG. Cleavage of the peptide bonds in this area of the molecule also reduced biopotency and immunochemical recognition by monoclonal antibodies directed to the heterodimeric hormone [12–14,18,19]. Based on known preferential cleavage sites in loop 2 several oligopeptides were synthesized, including MTR, MTRV, LQG, LQGV, VLPALP and VLPALPQ. In addition, the peptide VVC from the flanking COOH-side and the alanine substitution variant AQGV were synthesized. In the current study the eight synthetic oligopeptides were tested for their suppressive effect on LPS-induced septic shock in mice (Table 2). The results showed that all mice that were treated at 2 h post-LPS with LQGV, VLPALP or AQGV survived beyond 48 h, while all mice in the saline-treated control group had already succumbed from fatal septic shock within 24 h. Remarkably, the mice could even be rescued from fatal septic shock when treatment with either one of the oligopeptides MTR, MTRV, VLPALPQ, VVC or AQGV was started as late as 24 h post-LPS, with sickness scores higher than 4·0 (Table 2). Of the evaluated oligopeptides, only AQGV was fully effective in reducing the severity and mortality of septic shock at early (i.e. 2 h post-LPS) as well as late (i.e. 24 h post-LPS) administration. The different in vivo efficacy of AQGV and LQG was mirrored by a different in vitro immunosuppressive capacity. While AQGV reduced the in vitro proliferative response of splenocytes to LPS as well as CD3/IL-2, LQGV only reduced the proliferative response of splenocytes to CD3/IL-2. These data suggest that the suppressive effect of AQGV targets macrophages/monocytes, B cells and T cells and therefore possibly affects early and late mechanisms of septic shock in which different cell types and pathological pathways are active [20,21]. Although treatment with LQG, LQGV or VLPALP (5 mg/kg) at 24 h after LPS administration failed to reduce mortality, VLPALP reduced mortality to around 50% after administration of a threefold higher dose (15 mg/kg) at 24 h post-LPS [7]. This shows that detailed dose–response studies are needed for a full insight into the ability of the various hCG-related oligopeptides to inhibit LPS-induced shock in mice early and/or late after LPS administration.

Based on the observed beneficial effects of hCG-derived oligopeptides in the mouse septic shock model we chose to evaluate this new treatment principle in a septic shock model in deeply sedated rhesus monkeys induced by infusion of live E. coli. We selected the three most effective peptides in the mouse studies, namely AQGV, LQGV and VLPALP, and administered them as an i.v. cocktail. With regard to the primary end-point, all three monkeys that received mock treatment with saline succumbed from fatal septic shock within 8 h after the E. coli infusion. Four of five monkeys that were treated with the oligopeptide cocktail were protected completely against septic shock. Haemodynamic parameters, such as heart rate, blood pressure and SpO2, varied considerably between individual animals, but confirmed the clinical diagnosis of suppressed septic shock by oligopeptide treatment.

Two oligopeptide-treated monkeys without marked clinical symptoms were allowed to recover to assess whether the single treatment also protected them from late-onset shock symptoms. As one of these monkeys had to be killed with clinical signs of septic shock at 33 h post-E. coli, the second monkey received an additional infusion of the oligopeptide cocktail at 8 and 24 h post-E. coli. However, the additional two oligopeptide doses failed to rescue this monkey from multi-organ failure at 36 h, as could be concluded from the high serum levels of ASAT and ALAT, indicating liver damage, of creatinine and urea reflecting kidney dysfunction, as well as the non-selective markers LDH and lactate over the final 20 h (data not shown).

Evaluation of the secondary end-point, histological assessment of septic shock-related damage to vital organs, showed markedly less severe pathological changes in the three oligopeptide-treated monkeys that displayed no (Ri459) minor (Ri7046) or moderate clinical signs of septic shock (Ri11152) compared to the three saline-treated control monkeys. Despite the variability in primary and secondary end-point parameters that were obtained from a limited number of investigated cases, the overall data set warrants the conclusion that administration of an oligopeptide cocktail consisting of AQGV, LQGV and VLPALP protects rhesus monkeys against the clinical and pathological consequences of acute septic shock syndrome. Importantly, these results were established without any further supportive treatment. Despite the strong protective effect of the oligopeptide cocktail during the acute clinical crisis, monkeys that were allowed to recover from anaesthesia nevertheless succumbed within the following 24 h with clinical symptoms of septic shock. Moreover, at autopsy of the monkeys that were left alive and killed 1 day later with clinical signs of shock, the same pathological changes of septic shock syndrome as in the control monkeys could also be observed. This late complication of severe septic shock may be due to the translocation of bacteria from the damaged intestines.

To gain insight into the protective effect observed in the monkey model we measured TNF-α, IL-1β, IL-6 and IL-8 plasma levels. The plasma levels of these cytokines are elevated strongly during the initial 24 h after septic shock induction, and reduction of systemic levels and inhibition of these cytokines has been shown to be associated with improved survival in some animal models [22]. While we have observed that LQGV or AQGV treatment in a rat model of severe haemorrhagic shock reduced TNF-α and IL-6 serum levels [23], we observed similar plasma profiles for TNF-α, IL-1β, IL-6 and IL-8 in oligopeptide-treated and untreated rhesus monkeys. These data suggest that the oligopeptide treatment had no profound effect on the production or release of these proinflammatory cytokines in this model, suggesting that suppression of the systemic storm is not a probable explanation for the beneficial effect of the oligopeptide cocktail. However, the data on plasma cytokine levels should be interpreted with caution, as considerable variation was observed between individual monkeys. Moreover, the data were collected from a small number of monkeys used, which differed in genetic background and age. Hence, we cannot exclude completely the possibility that the oligopeptides affect systemic cytokine release in this model.

Gender-specific differences in immune responses after shock occur and have been attributed to the different immunomodulatory effects of male and female steroid hormones [24]. In our study we used non-pregnant female mice and monkeys, but did not check for the oestric cycle or female sex hormone levels. Nevertheless, we consider it unlikely that female sex hormones have influenced the different outcome between peptide and non-peptide treated animals, as these peptides have been found to inhibit haemorrhagic shock-associated inflammation in male rats [23].

Thus far it is unclear by which molecular mechanism the remarkable pleiotropic beneficial effects of hCG-related oligopeptides in different models of acute shock are mediated. Recently we reported that male rats treated with either LQGV or AQGV alone, at 30 min after the onset of severe haemorrhagic shock, displayed not only reduced plasma levels of TNF-α and IL-6, but also reduced E-selectin, TNF-α and IL-6 mRNA transcript levels in the liver. LQGV treatment was also associated with a significant reduction of neutrophil accumulation in the liver [23]. Endothelial activation, characterized by increased expression of adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)] and subsequent tissue infiltration by neutrophils is an early event in septic shock and a crucial step towards the development of organ failure [20,25,26]. E-selectin blockage has been shown to protect against neutrophil-induced tissue injury during endotoxaemia in mice [27]. Considering these data and our previous findings [23], we hypothesize that reduced E-selectin expression upon oligopeptide treatment may have contributed to the lower inflammatory cell accumulation and reduced organ damage, as was observed in the rhesus monkey study presented here.

Gene expression analysis in several rodent models of inflammatory disease confirmed that treatment with LQGV or AQGV reduces gene expression for proinflammatory cytokines and adhesion molecules (Khan et al. in preparation). This suggests that the immune regulatory effect of the hCG-related regulatory oligopeptides in rodents may, at least in part, be driven by the regulating gene expression. It is as yet unknown, however, whether the oligopeptides act by binding to surface-expressed or cytosolic receptors. Preliminary data exclude that LQGV, AQGV and VLPALP act via binding to the luteinizing hormone (LH) receptor as they do not bind to the LH receptor nor do they interfere with the binding of hCG to the LH receptor (data not shown). Due to their small size and low molecular weight, these oligopeptides might easily pass the cell membrane [28] and interfere with signalling cascades or interact with regulatory sequences or transcriptional complexes.

In conclusion, the results of the current study show marked beneficial effects of some synthetic oligopeptides (3–7 amino acids) related to the primary structure of the pregnancy hormone hCG in experimental models of acute septic shock in two different species. No adverse effects of the hCG-related oligopeptides were observed in these two species. Therefore these oligopeptides may have therapeutic value for this often-fatal condition.

A Phase Ia multi-dose safety trial with AQGV (EA-230) in man has been completed [29], in which subjects received three times daily i.v. infusions of EA-230 for 3 days. No significant adverse events were found to be associated with the use of EA-230. Also a Phase Ib trial – designed as a double-blind, randomized, single-dose, placebo-controlled LPS challenge trial with EA-230 – has been completed [30]. The primary aim of this trial was to determine whether EA-230 administration attenuates the inflammatory response induced by LPS infusion into healthy volunteers. In the trial one group each of 12 subjects received LPS by injection, followed 30 min later by either administration of EA-230 or placebo. Pharmacokinetic analysis of the study subjects showed not just a reduction of blood C-reactive protein levels, but also reduction of several proinflammatory cytokines (IL-6, IL-8 and TNF-α) compared to the placebo group. In addition, the treatment also reduced the rise of body temperature and the drop in white blood cell counts that are induced typically by LPS. Correspondingly, the data revealed an increase in the anti-inflammatory cytokine IL-10. The outcome of these studies has been considered successful, because they suggest that EA-230 may reduce a systemic inflammatory response elicited by endotoxin. This LPS study was selected to create an artificial proxy for an actual clinical scenario. These study results, combined with the results of Phase Ia single-dose trials, provide the basis for the development and implementation of more comprehensive Phase II human trials on EA-230.

Acknowledgements

We thank our colleagues of the Animal Science Department of the BPRC, in particular Mr Leo van Geest, veterinary assistant, for expert experimental support. This study was financially supported via an unbiased grant from Biotempt BV, Koekange, the Netherlands.

Disclosure

None.