Clinical Chemistry Profiles Research Articles

Safety of AYUSH OSTO in experimental animals

Background: AYUSH OSTO is a coded formulation developed by the Central Council for Research in Ayurvedic Sciences for the treatment of bone-related disorders. Aim: The present study focused on evaluating the safety of AYUSH OSTO through acute, 28-day sub-acute, and 90-day sub-chronic repeated dose toxicity studies. Materials and Methods: Acute oral toxicity was tested with doses of 260, 1300, and 2600 mg/kg, and sub-acute, sub-chronic studies were performed with doses of 180, 900, and 1800 mg/kg. During the acute toxicity, the evaluation test compound was administered once orally, whereas in the sub-acute toxicity and sub-chronic studies, the test compound was administered daily for 28 days and 90 days, respectively. Clinical signs of toxicity and mortality were observed in all three studies. Feed intake, changes in body weight, hematological, clinical biochemistry, urine analysis, gross necropsy, and histopathological analysis were performed in sub-acute, sub-chronic studies. Results: There was no mortality, and no clinical signs of toxicity were observed in all three toxicity studies. The weekly feed intake and body weight changes were found to be nonsignificant when compared to the control. The clinical chemistry and hematological profiles of the animals were not altered with the treatment. The histopathological analysis of vital organs at the tested dose levels did not show any organ damage. Conclusions: The findings of the acute toxicity study established the approximate LD50 of >2600 mg/kg and the repeated dose toxicity studies established the no-observed-adverse-effect level of AYUSH OSTO is up to 1800 mg/kg.

Effects of fermented Crescentia cujete L. on the profile of hematology, clinical chemistry, and circulatory CD4+/CD8+ in Sprague Dawley rats.

The calabash (Crescentia cujete L.) is a tropical fruit that offers numerous health benefits. Its fermented calabash (FC) has been found to affect the neurological system positively. However, its impact on hematology, clinical chemistry, and CD4+ and CD8+ levels has yet to be documented. Therefore, this study aims to examine the effects of FC on hematology, clinical chemistry, and the levels of CD4+ and CD8+ in the circulatory system using rat models. This study used twenty male Sprague Dawley rats. The rats were divided into group 1 (0 mg/kg BW FC), group 2 (50 mg/kg BW FC), group 3 (500 mg/kg BW FC), and group 4 (2,000 mg/kg BW FC). The treatment was administered using a gastric probe once daily for 14 days. On day 15, the blood samples were collected and tested against hematology, clinical chemistry, quality of the erythrocytes, and CD4+/CD8+. The data were then analysed using SPSS with p-value at 0.05. The conducted study demonstrated that the utilization of FC at varying doses did not have a significant impact on the hematological profile changes (p ≥ 0.05), except for total lymphocytes and a decrease in the neutrophils/lymphocytes (N/L) ratio (p ≤ 0.05). Furthermore, FC did not influence the changes in clinical chemistry, circulatory protein, and electrolyte levels in rat models compared to the control (p ≥ 0.05). The utilization of FC decreased the percentage of hemolysis and elevated the adenosine triphosphate (ATP) concentration (p ≤ 0.05). Additionally, the use of FC led to a significant increase in CD4+ and the ratio of CD4+/CD8+ (p ≤ 0.05), while no significant effect was observed regarding CD8+ (p ≥ 0.05). The study highlighted FC's beneficial effects on the haemorheology and immune system, specifically on the decrease in the percentage of hemolysis, elevated ATP concentration, number of lymphocytes, ratio N/L, CD4+, and the CD4+/CD8+ ratio, without causing significant changes to the hematological and clinical chemistry profiles in rat models.

Impaired flux of bile acids from the liver to the gut reveals microbiome-immune interactions associated with liver damage

Currently, there is evidence that alteration in the gut ecosystem contributes to the development of liver diseases, however, the complex mechanisms involved are still unclear. We induced cholestasis in mice by bile duct ligation (BDL), mirroring the phenotype of a bile duct obstruction, to understand how gut microbiota alterations caused by an impaired flow of bile acid to the gut contribute to the pathogenesis and progression of liver disease. We performed longitudinal stool, heart, and liver sampling using mice receiving BDL and controls receiving sham operation (ShamOP). Shotgun metagenomics profiling using fecal samples taken before and on day 1, day 3, and day 7 after surgery was performed, and the cytokines and clinical chemistry profiles from heart blood, as well as the liver bile acids profile, were measured. The BDL surgery reshaped the microbiome of mice, resulting in highly distinct characteristics compared to the ShamOP. Our analysis of the microbiome pathways and ECs revealed that BDL reduces the production of hepatoprotective compounds in the gut, such as biotin, spermidine, arginine, and ornithine, which were negatively associated with inflammatory cytokines (IL-6, IL-23, MCP-1). The reduction of the functional potential of the gut microbiota in producing those hepatoprotective compounds is associated with the decrease of beneficial bacteria species from Anaerotruncus, Blautia, Eubacterium, and Lachnoclostridium genera, as well as the increase of disease-associated bacteria e.g., Escherichia coli and Entercoccus faecalis. Our findings advances our knowledge of the gut microbiome-bile acids-liver triangle, which may serve as a potential therapeutic strategy for liver diseases.

Acute and Subacute Toxicity of Rhamnus prinoides Leaves on Histopathology of Liver, Kidney, and Brain Tissues, and Biochemical Profile of Rats.

Rhamnus prinoides is used as a traditional medicinal plant to treat pneumonia, sprain, gonorrhea, rheumatism, and ringworm infections as well as for the preparation of local beverages in Ethiopia. It has a widespread antioxidant, antimalarial, antimicrobial, wound healing, and anti-inflammatory activities. These activities are due to the presence of alkaloids, steroids, triterpenes, tannins, flavonoids, flavones, phenols, and glycosides. This study aimed to investigate acute and subacute toxicity of R. prinoides leaves on histopathology of the liver, kidney, and brain tissues, and biochemical profiles of rats. For the acute toxicity study, female rats were treated with R. prinoides at a dose of 5000 mg/kg body weight and followed-up for 14 days. In the subacute toxicity study, four groups of rats were used. The first three groups, respectively, received 250, 500, and 1000 mg/kg body weight of R. prinoides extract and the fourth group was a control group. Signs of toxicity, food intake, and weight was recorded. At necropsy, organ weight measurement and macroscopic and microscopic evaluations of the liver, kidney, and brain were carried out. Different clinical chemistry profiles of rats were also measured. Single-dose oral administration of R. prinoides extract at 5000 mg/kg produced no mortality indicating the LD50 is greater than 5000 mg/kg body weight. A four week administration of R. prinoides extract did not bring deleterious outcomes on the food consumption and weight gain of rats. Moreover, gross examination, histopathological evaluation, and weight measurement conducted on the liver, kidney, and brain did not reveal treatment related changes. The biochemical analysis showed no significant difference between the treatment and control groups. Consumption of R. prinoides leaf for 4 weeks might not have a toxic effect in rats. However, further investigations upon long-term administration should be conducted to have a wider safety margin.

Clinical Development of Senza5TM CART5: a Novel Dual Population CD5 CRISPR-Cas9 Knocked out Anti-CD5 Chimeric Antigen Receptor T Cell Product for Relapsed and Refractory CD5+ Nodal T-Cell Lymphomas

Introduction: Chimeric antigen receptor T-cell (CART) immunotherapy is FDA-approved for B-cell lymphoblastic leukemia, B-cell non-Hodgkin lymphomas, and multiple myeloma. However, CART immunotherapy for T-cell non-Hodgkin lymphomas (T-NHL) is lagging, with only limited clinical results. T-NHL have a poor prognosis and there is an unmet need for effective and long-lasting treatments. We previously presented preliminary preclinical results of anti-CD5 CAR T cells (CART5) that had CD5 knocked out (KO) (Blood (2020) 136 (Supplement 1): 51-52.). CD5 KO not only prevented CART fratricide but also dramatically enhanced CART function. Critical challenges to developing CART cells for T-NHL are CART fratricide and normal T-cell toxicity. In this study, we addressed these challenges by improving manufacturing and including a dual-population technology to mitigate toxicity. We present the development of Senza5TM CART5 an autologous good-manufacturing practice (GMP)-grade product (Fig.1) that will be tested in a phase I clinical trial for relapsed and refractory CD5+ nodal T-cell lymphomas. Methods and Results: We first identified a single guide RNA (sgRNA) that deletes CD5 in T cells with >90% KO efficiency by flow cytometry and ICE analysis. We then assessed the specificity of the KO by performing iGUIDE sequencing. We confirmed high on-target CD5 cleavage with minimal off-targets (CALCP, C20orf85, INPP4B, XPO7, and SLC10A7). These genes have little to no expression in naïve CD4+ and CD8+ T cells by RNA-seq (DICE). Targeted functional assays are ongoing. For the CAR construct, we selected a lead CAR5 among six 4-1BB-constructs based on its superior anti-tumor function in NOD-SCID IL2Rgnull (NSG) mice engrafted with a Jurkat T-lymphoblastic leukemia cell line. Furthermore, to confirm the specificity of the CAR5 for CD5, we screened CAR5 T cells for interactions with HEK293 cells expressing 5,828 human plasma membrane proteins or cell-surface tethered secreted proteins and 398 human heterodimers (Retrogenix assay). The lead CAR5 did not bind to any targets besides CD5. For an optimized GMP-grade product, we modified our manufacturing protocol, shortening it from 10 to 5 days. Moreover, KO efficiency and safety were optimized to reduce off-target activity without loss of efficiency. We also optimized the activation strategy to decrease cell loss. Functional studies were performed to demonstrate the comparability of these methods. With these improvements, we were able to achieve >90% CD5 KO with >30% CAR transduction, thereby allowing the generation of two main populations: one of CD5KO CART5 cells and the other of CD5KO normal untransduced T cells. CD5KO CART5 are expected to target CD5+ T-NHL cells but could also lead to normal T-cell toxicity. This toxicity will be mitigated by the CD5KO normal T cells that will have a survival advantage after infusion (Fig.1) and have been demonstrated to maintain reactivity against opportunistic pathogens such as CMV and EBV. To evaluate Senza5TMCART5 safety, we performed cytokine-independent CART proliferation and long- and short-term xenograft models. Senza5TM CART5 cells did not grow aberrantly without growth/survival-associated cytokines (IL2/7/15). We then used NSG mice to exclude uncontrolled Senza5TM CART5 growth and observed enhanced expansion and proliferation of Senza5TM CART5 with a xenogeneic GVHD rate similar to that of control mice with similar wild-type CART5 expansion. Since the CAR5 does not cross-react with murine CD5, we tested non-specific toxicity in tissues using an NSG xenograft model of Jurkat. On Day 10 post-CAR T injections, animals were euthanized, and a complete necropsy was performed on all animals. Senza5TM CART5 demonstrated tumor clearance with no detection of treatment-related lesions. Mice treated with untransduced T cells or CART19 demonstrated significant overall and organ-specific weight loss compared to Senza5TM CART5. Blood analyses determined no differences in their clinical chemistry profile or complete blood counts. Conclusion: In this study, we developed a novel dual-population, full-scale GMP-ready CD5 KO anti-CD5 CART product for T-cell lymphomas characterized by enhanced anti-tumor effect and a strategy to potentially mitigate toxicity. Autologous Senza5TM CART5 will be tested for CD5+ nodal T-cell lymphomas in a phase I clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nonclinical safety evaluation of oral recombinant anti-human papilloma virus vaccine (RHPV 16 & 18): Regulatory toxicology studies in mice, rats and rabbits – An innovative approach

AimThe human papilloma virus (HPV) type 16 and 18 causes nearly 70% of uterine cervical cancers. Oral administration of live Salmonella typhi Ty21a, expressing major capsid proteins (L1) of HPV 16 and 18 is a potential choice for immunization in adolescent girls under low resource settings. Present study aimed to assess the nonclinical safety of recombinant S. typhi expressing HPV 16 and 18 (rStHPV) proteins. MethodologyThe acute toxicity of rStHPV was tested by intranasal single dose administration, of 10 and 50 folds higher than clinical prophylactic dose, in mice and rat followed by monitoring their survival for 14 days. Sub-chronic toxicity was evaluated in rats and rabbits with prophylactic and 5 times (average) to clinical prophylactic dosages on scheduled days (1st, 3rd & 5th day) through oral and intranasal routes. The immune/allergic response of rStHPV was assessed in mice through intranasal and intra-peritoneal routes. Experimental animals were daily monitored for live phase, and clinical chemistry, haematology, immunotoxicology, immunogenic response and histopathological examination of vital organs on 15th, 29th and 93rd days. ResultsNo abnormal changes were noticed in live phase activity, clinical chemistry and haematology profile. The gross necropsy, organ weights and histopathology were found to be normal. No immunotoxicity was recorded as evaluated by tier I tests. Allergic immune response, as evaluated with IgE levels was also negative irrespective of test routes. On the other hand, a significant (P < 0.01) increase of anti-HPV IgG levels was noted in mice exposed through intranasal route. Though the pre-terminal mortality was noted in mice (6–15%), rats (10%) and rabbits (15%), the autopsy revealed no signs of toxicity related to rStHPV, as the changes neither significant nor dose dependent; and even noted in vehicle control also. ConclusionThe study results suggested ‘no observable adverse effects’ of rStHPV even at higher dosages (5, 10 & 50 folds) than intended clinical dose. A significant increase of anti-HPV specific IgG suggests the immunogenicity of vaccine. The innovative approach of current study is nonclinical toxicology evaluation of vaccine through intra-nasal route, an alternate route apart from stipulated regulatory guidelines.

Efficacy of semi-annual therapy of an extended-release injectable moxidectin suspension and oral doxycycline in Dirofilaria immitis naturally infected dogs

BackgroundDirofilaria immitis is a life-threatening nematode spreading globally. Arsenical treatment is currently recommended for removal of adult worms. However, arsenical treatment is not available in some countries, and there are dogs that cannot tolerate the rapid kill of adult worms; therefore, alternative adulticide slow-kill treatments are needed. Criticisms against the use of these alternative protocols include the potential for allowing disease to progress and for the development of ML-resistant worms.MethodsThe efficacy of a protocol that includes semi-annual doses (i.e. every 6 months) of commercially available extended-release injectable moxidectin suspension (ProHeart® SR-12) with 30-day oral administration of doxycycline was studied in 20 dogs with naturally occurring D. immitis infections. Each dog received treatment with ProHeart® SR-12 (0.5 mg moxidectin/kg) by subcutaneous injection and oral doxycycline (10 mg/kg/bid × 30 days) every 6 months until two consecutive negative antigen test results were obtained. Pulmonary and cardiac evaluations were performed by radiographic and echocardiographic parameters. Physical examinations, complete blood counts, clinical chemistry profiles, microfilariae and antigen tests were performed periodically.ResultsAt enrollment, all dogs were positive for D. immitis antigen and 18 were microfilaremic. On day 30, microfilaremia counts decreased, and all dogs became amicrofilaremic by day 150. On day 180, 11 dogs were antigen-negative, and 7 more became negative by day 360. The two remaining antigen-positive dogs converted to negative by day 540 or 810. All antigen tests performed 180 days after the first negative test were negative. There was no decline in cardiac performance of the dogs throughout the study. Overall, pulmonary clinical conditions, presence of worms by echocardiography, and enlargement of caudal and main pulmonary arteries improved after treatment. Physical examinations, complete blood count results, and clinical chemistry profiles were within normal reference values. Respiratory conditions were improved, no damage to the heart was observed, and the treatment protocol was well tolerated by the animals.ConclusionsThis alternative adulticide treatment was efficacious and well tolerated in naturally infected dogs. The injectable formulation provides the advantage of having veterinarians able to administer, monitor, and assess the efficacy and condition of the dog throughout the treatment and post-treatment periods.

Preliminary assessment of the new Sysmex XN parameter Iron-Def for identifying iron deficiency.

Although the clinical assessment of iron status is usually based on iron stores, a rapid and accurate diagnosis of iron deficiency is challenging since ferritin is often unavailable as an urgent test and its value is frequently increased in acute phase conditions. This study was therefore aimed at evaluating the diagnostic performance of the new Sysmex XN "Iron Deficiency?" (Iron-Def) parameter for identifying patients with iron deficiency. The study population consisted of 688 consecutive patients (median age: 71 years; 341 women and 347 men), referred for routine diagnostics to the Laboratory of Clinical Pathology of Lecco Hospital, Italy. A complete clinical chemistry profile and haematological testing were performed for identifying iron deficiency anaemia. A significant negative correlation was found between Sysmex XN Iron-Def and ferritin, serum iron, mean cell haemoglobin concentration, mean cell haemoglobin, mean corpuscular volume and age, while a positive correlation was noted with transferrin, percentage of microcytic red cell, red blood cell count and red blood cell distribution width. The diagnostic accuracy of Iron-Def for identifying patients with a percentage of saturation of transferrin <15% (n=104) was 84%, with a sensitivity of 0.952 and specificity of 0.538. A sub-analysis of 71 patients with ferritin <20 ng/dL yielded an even better diagnostic performance (86%, with a sensitivity of 0.935 and specificity of 0.620). Although additional confirmatory investigations would be needed, the preliminary findings of our study attest that Iron-Def may be an easy, inexpensive, rapid and reliable parameter for screening iron deficiency anaemia.

Follow-up study in German Hunting Terrier dogs with exercise induced metabolic myopathy

Exercise induced metabolic myopathy in German Hunting Terrier dogs is an autosomal-recessively inherited disorder, caused by a nonsense variant of the gene encoding for the very long-chain acyl-CoA-dehydrogenase (VLCAD) enzyme. Clinical signs include exercise- induced fatigue, muscle pain and weakness. In the present study, the long-term course of this disease was investigated over a period of 1 year in 9 affected German Hunting Terriers. The dogs were treated symptomatically with oral L-carnitine, coenzyme Q10 and a special diet characterized by a low content of long-chain fatty acids and a high proportion of carbohydrates. In 9 affected dogs, the phenotype as well as clinical, laboratory parameters, and histopathological findings are described (time point 1) and compared to follow-up examinations 1 year later (time point 2). At both time points clinical and neurological examinations, complete blood cell count, clinical chemistry profile and the concentration of brain natriuretic peptide (NT-proBNP) were investigated. In the follow-up examinations, the same post-exercise clinical signs were present as in the initial presentation of the homozygous dogs. Dark-brownish discoloration of the urine, weakness, myalgia as well as stiff and tetraparetic gait were apparant. All hematological values and the concentration of NT-proBNP were within the relevant reference ranges. Plasma CK and ALT activities were compared between the first presentation and the follow- up examination and no significant differences were detected (pCK = 0.31, pALT = 0.64). Signs of myopathy remained unchanged throughout the examination period. Oral supplementation with L-carnitine, coenzyme Q10 and the special dietary management did not result in any improvement of clinical signs or laboratory parameters. No progression of the disease was observed. The prognosis for affected dogs remains cautious as long-term observations of affected dogs over several years are lacking. Our findings provide further important information on inherited disorders of mitochondrial β-oxidation in dogs, especially focused on the exercise induced metabolic myopathy in the German Hunting Terrier. This may provide new insights for novel treatment modalities in conjuntion with the development of improved breeding guidelines.

Exploring Covariation between Traditional Markers of Metabolic Health and the Plasma Metabolomic Profile: A Classic Twin Design.

Novel metabolomic profiling techniques combined with traditional biomarkers provide knowledge of mechanisms underlying metabolic health. Twin studies describe the impact of genes and environment on variation in traits. This study aims to identify relationships between traditional markers of metabolic health and the plasma metabolomic profile using a twin modeling approach and determine whether covariation is caused by shared genetic and environmental factors. Using a classic twin design, this study examined covariation between anthropometric, clinical chemistry, and metabolomic profiles. Cholesky decomposition modeling was used to determine the genetic and environmental path coefficients through successive anthropometric and clinical chemistry traits onto metabolomic derived metabolites. This study shows that WC, TAG, and a metabolomic signature composed of 7 metabolites are inter-related, and that covariation can be attributed to common genetic, shared and unique environmental factors as well as unique environmental factors specific to the metabolite. This quantitative modeling connecting the traditional anthropometry and clinical chemistry traits with the more recent and potentially more sensitive metabolomic profile approach may provide further insight on the pleiotropic genes or modifiable environmental factors influencing variation in metabolic health.

Impact of nitinol stent surface processing on in-vivo nickel release and biological response

Although nitinol is widely used in percutaneous cardiovascular interventions, a causal relationship between nickel released from implanted cardiovascular devices and adverse systemic or local biological responses has not been established. The objective of this study was to investigate the relationship between nitinol surface processing, in-vivo nickel release, and biocompatibility. Nitinol stents manufactured using select surface treatments were implanted into the iliac arteries of minipigs for 6 months. Clinical chemistry profile, complete blood count, serum and urine nickel analyses were performed periodically during the implantation period. After explant, stented arteries were either digested and analyzed for local nickel concentration or fixed and sectioned for histopathological analysis of stenosis and inflammation within the artery. The results indicated that markers for liver and kidney function were not different than baseline values throughout 180 days of implantation regardless of surface finish. In addition, white blood cell, red blood cell, and platelet counts were similar to baseline values for all surface finishes. Systemic nickel concentrations in serum and urine were not significantly different between processing groups and comparable to baseline values during 180 days of implantation. However, stents with non-optimized surface finishing had significantly greater nickel levels in the surrounding artery compared to polished stents. These stents had increased stenosis with potential for local inflammation compared to polished stents. These findings demonstrate that proper polishing of nitinol surfaces can reduce in-vivo nickel release locally, which may aid in minimizing adverse inflammatory reactions and restenosis. Statement of significanceNitinol is a commonly used material in cardiovascular medical devices. However, relationships between nitinol surface finishing, in-vivo metal ion release, and adverse biological responses have yet to be established. We addressed this knowledge gap by implanting single and overlapped nitinol stents with different surface finishes to assess systemic impact on minipigs (i.e. serum and urine nickel levels, liver and kidney function, immune and blood count) over the 6 month implantation period. In addition, nickel levels and histopathology in stented arteries were analyzed on explant to determine relationships between surface processing and local adverse tissue reactions. The findings presented here highlight the importance of surface processing on in-vivo nickel release and subsequent impact on local biological response for nitinol implants.

Mycoprotein represents a bioavailable and insulinotropic non-animal-derived dietary protein source: a dose-response study.

The anabolic potential of a dietary protein is determined by its ability to elicit postprandial rises in circulating essential amino acids and insulin. Minimal data exist regarding the bioavailability and insulinotropic effects of non-animal-derived protein sources. Mycoprotein is a sustainable and rich source of non-animal-derived dietary protein. We investigated the impact of mycoprotein ingestion, in a dose-response manner, on acute postprandial hyperaminoacidaemia and hyperinsulinaemia. In all, twelve healthy young men completed five experimental trials in a randomised, single-blind, cross-over design. During each trial, volunteers consumed a test drink containing either 20 g milk protein (MLK20) or a mass matched (not protein matched due to the fibre content) bolus of mycoprotein (20 g; MYC20), a protein matched bolus of mycoprotein (40 g; MYC40), 60 g (MYC60) or 80 g (MYC80) mycoprotein. Circulating amino acid, insulin and uric acid concentrations, and clinical chemistry profiles, were assessed in arterialised venous blood samples during a 4-h postprandial period. Mycoprotein ingestion resulted in slower but more sustained hyperinsulinaemia and hyperaminoacidaemia compared with milk when protein matched, with overall bioavailability equivalent between conditions (P>0·05). Increasing the dose of mycoprotein amplified these effects, with some evidence of a plateau at 60-80 g. Peak postprandial leucine concentrations were 201 (sem 24) (30 min), 118 (sem 10) (90 min), 150 (sem 14) (90 min), 173 (sem 23) (45 min) and 201 (sem 21 (90 min) µmol/l for MLK20, MYC20, MYC40, MYC60 and MYC80, respectively. Mycoprotein represents a bioavailable and insulinotropic dietary protein source. Consequently, mycoprotein may be a useful source of dietary protein to stimulate muscle protein synthesis rates.

Assessment of parasitological findings in heartworm-infected beagles treated with Advantage Multi\xae for dogs (10% imidacloprid\u2009+\u20092.5% moxidectin) and doxycycline

BackgroundAnecdotal reports support the position that the adulticidal heartworm treatment utilizing doxycycline and Advantage Multi®/Advocate® for Dogs (10% imidacloprid + 2.5% moxidectin) has successfully converted antigen-positive dogs to antigen-negative. To date, no controlled experimental studies have demonstrated the adulticidal efficacy of this treatment regimen. The aim of this study was to evaluate the parasitological and clinical efficacy of Advantage Multi® for Dogs (IMD + MOX) and doxycycline in heartworm-infected beagles.MethodsThis study utilized 16 dogs, 8 dogs in each of non-treated control and treated groups. A total of 16 adult Dirofilaria immitis (Missouri strain) were surgically transplanted into the jugular vein of each study dog. The treatment regimen of monthly IMD + MOX topically (per labeled dosage and administration) for 10 months and 10 mg/kg doxycycline BID orally for 30 days was initiated 30 days post-surgical transplant. Echocardiograms, radiographs, complete blood counts, clinical chemistry profiles, heartworm antigenemia and microfilaremia were evaluated every 4 weeks. Serum samples were assayed for heartworm antigen using the DiroCHEK® heartworm antigen test. The DiroCHEK® was performed according to the manufacturer’s recommendations and read using a spectrophotometer at 490 nm.ResultsAll dogs tested positive for the presence of heartworm antigen post-surgical transplant and prior to treatment. Heartworm antigen levels began declining in treated dogs 3 months post-treatment. Non-treated control dogs remained antigen-positive. No microfilariae were detected in treated dogs after 21 days post-treatment. At necropsy, adult heartworms were recovered from all non-treated control dogs with a range of 10–12 adult worms/dog for an average recovery of 10.6 adult heartworms/dog. In the IMD + MOX- and doxycycline-treated dogs, the range of adult heartworms recovered was 0–2 adult worms/dog, with five dogs having no adult heartworms present. The average adult heartworm recovery was 0.6/dog in the treated group. This treatment regimen demonstrated a 95.9% efficacy in eliminating adult heartworms (P < 0.0001).ConclusionsThis study demonstrated that this treatment regimen successfully eliminated D. immitis microfilariae by 21 days post-treatment, reduced heartworm antigen concentration over time, and had a 95.9% efficacy in the elimination of mature adult heartworms. Based on this study, we conclude that this treatment regimen is a relatively quick, reliable and safe option to treat canine heartworm infection as compared to other treatment regimens involving macrocyclic lactones, when the approved drug melarsomine dihydrochloride is unavailable, contraindicated or declined by an owner unable to afford the more costly treatment or concerned about the potential side effects.

Subchronic toxicity studies of aqueous leaf and stem bark extract of Irvingia gabonensis in male albino wistar rats

Irvingia gabonensis is a plant with enormous ethno-botanical profile. It is largely used in traditional and modern medicine for the treatment of several illnesses. This study investigated the effect of aqueous leaf and stem bark extracts of I. gabonensis on the haematology, pathomorphology and some clinical chemistry profiles of male albino wistar rats following subchronic exposure. The extract was administered using the oral route. Group A served as control and was given only distilled water. Groups B, C and D were treated with the extract at 100, 1000 and 2000 mg/kg, respectively. The parameters assessed to determine the effect of subchronic administration of the extract were: packed cell volume (PCV), haemoglobin concentration (Hb), total red blood cell count (TRBC), total white blood cell count (TWBC), differential white blood cell count (DWBC), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) and histopathology of the heart, liver, spleen, kidney and testes. The data obtained were subjected to one-way analysis of variance (ANOVA). Values were considered statistically significant at p < 0.05. The haematological values obtained from the treatment groups did not show any significant difference from the control. Likewise, the serum biochemistry values obtained from the treatment groups did not differ significantly from the control. Histopathological evaluation of the testes showed testicular degeneration in groups C (1000 mg/kg) and D (2000 mg/kg). The findings of this study suggest that I. gabonensis may have a reprotoxic effect following subchronic or long-term exposures.

Clinical Manifestations, Hematology, and Chemistry Profiles of the Six Most Common Etiologies from an Observational Study of Acute Febrile Illness in Indonesia

Background Infectious diseases remain a significant healthcare burden in the developing world. In Indonesia, clinicians often manage and treat patients solely based on clinical presentations since the diagnostic testing capacities of hospitals are limited. Unfortunately, the most common infections in this tropical environment share highly similar manifestations, complicating the identification of etiologies and leading to the misdiagnosis of illness. When pathogen-specific testing is available, generally at top-tier specialist hospitals, the limited range of tests and slow turnaround times may never lead to a definitive diagnosis or improved patient outcomes.Methods To identify clinical parameters that can be used for differentiating the most common causes of fever in Indonesia, we evaluated clinical data from 1,486 acute febrile patients enrolled in a multi-site observational cohort study during 2013 to 2016.ResultsFrom the 66% of subjects with confirmed etiologies, the six most common infections were dengue virus (455), Salmonella spp. (124), Rickettsia spp. (109), influenza virus (64), Leptospira spp. (53), and chikungunya virus (37). The accompanying figure shows the clinical signs and symptoms (A) and hematology and blood chemistry results (B) for the color-coded pathogens. Comparing the profiles of all infected subjects reveals parameters that are uniquely associated with particular pathogens, such as leukopenia with dengue virus.Conclusion These observations will assist clinicians in healthcare systems with limited diagnostic testing capacities and may be useful in formulating diagnostic algorithms for Indonesia and other developing countries.Disclosures All authors: No reported disclosures.

Safety evaluation of tangeretin and the effect of using emulsion-based delivery system: Oral acute and 28-day sub-acute toxicity study using mice

Polymethoxyflavones, found widely in the peel of citrus fruits, is an emerging group of bioactive compounds with wide arrays of disease prevention functionalities. To understand the potential oral toxicity, tangeretin, being one of the most abundant polymethoxyflavones from natural sources, was used as model compound for the safety evaluation. Acute oral toxicity study was conducted using both male and female mice giving 1000, 2000, or 3000mg/kgbody weight (bw) of tangeretin in oil suspension from single gavage administration. No evidence of death was observed during 14-day post-administration period. Alterations of the hepatic cell and clinical chemistry profile increased dose dependently and exhibited distinct injury recovery pattern among different sexes. To determine the potential safety concern related to emulsification, the sub-acute toxicity of tangeretin in emulsion was evaluated and compared with un-processed oil suspension when conducting the sub-acute toxicity study over 28days. In the sub-acute study, emulsion system did not induce a significant increase of toxicity response. However, the daily low-dose application of tangeretin showed U-shaped dose–response pattern in regard to hepatic alteration. The result from this study can serve as a good safety reference for future application of polymethoxyflavone as a functional ingredient in food.

Safety of a Bioactive Polyphenol Dietary Supplement in Pediatric Subjects with Acute Diarrhoea.

The hematological and clinical chemistry profile for children aged 6 months to 5 years with acute diarrhoea was measured in a double blind clinical trial. Subjects were randomized to the study group (N = 44) given a bioactive polyphenol dietary supplement in oral rehydration solution (ORS) or to the control group (N = 41) given distilled water as a placebo in ORS twice daily for up to 4 days. All subjects received 10 mg zinc daily for the 4 days in the study. Venous blood was collected for complete blood count, electrolytes, liver function, and creatinine upon enrollment (baseline) and at the end of 4 days (end of study); mean values were compared by 95% confidence intervals. Overall, blood factors measured either remained the same over the 4 days or increased or decreased at the same levels between the two groups during the study period. All values were within accepted ranges for paediatric subjects except serum AST (SGOT), where the mean value of the study group approached the upper bound of the range on day 4 but was comparable to the value of the control group. Consumption of this supplement twice daily for 4 days is safe for children and infants.

Heartworm-positive dogs recover without complications from surgical sterilization using cardiovascular sparing anesthesia protocol

Expedited home placement of heartworm-positive shelter dogs is desirable and may outweigh the perceived increase in anesthetic risk for pre-adoption sterilization; however, this issue has not been addressed in the literature. Our goal was to determine whether heartworm-positive dogs suffered clinically evident perioperative complications after sterilization under general anesthesia. Anticipated complications could result from anesthesia-induced changes in pulmonary vascular resistance and cardiac output leading to signs of thromboembolic disease and even death from dislodged parasites. The medical records of 15 hemodynamically stable, intact, heartworm-positive, mixed-breed shelter dogs with no or mild clinical signs were examined. Pre-operative evaluation of patients included a complete blood count, clinical chemistry profile, heartworm antigen and microfilariae screen, electrocardiogram, and thoracic radiographs. The anesthetic protocol for heartworm-positive dogs included acepromazine (0.01–0.05mg/kg), butorphanol (0.1mg/kg IM) and meloxicam (0.2mg/kg IM), or carprofen (2mg/kg SQ) in the preanesthetic period; tiletamine/zolazepam (3–6mg/kg IV) or ketamine/diazepam (3–6mg/kg/0.25–5mg/kg IV) to effect for induction; maintenance on isoflurane or sevoflurane and oxygen. A lidocaine testicular block was performed on 11 males. All dogs were monitored postoperatively for a minimum of 24h and then daily until discharge. There were no clinically evident perioperative complications in heartworm-positive dogs. Purposeful pre-operative evaluation of heartworm-positive dogs while utilizing cardiovascular-sparing anesthetic protocols may allow clinicians to proceed with sterilization of hemodynamically stable heartworm-positive shelter dogs prior to heartworm treatment.

Can the use of blood-based biomarkers in addition to anthropometric indices substantially improve the prediction of visceral fat volume as measured by magnetic resonance imaging?

To investigate whether blood-based biomarkers can improve the prediction of visceral fat volume as measured by magnetic resonance imaging (MRI) and thus be used as proxies of visceral adiposity in large-scale epidemiological studies. Whole-body MRI was performed to determine overall and regional body compartments in 542 participants aged 48-80 years (52% men) of the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition. Anthropometric measures were taken, and clinical chemistry profiles including 15 routine biomarkers were obtained. Furthermore, nine novel biomarkers of visceral fat were assayed in a discovery sample of 100 participants. Multivariable regression models were calculated to assess associations between anthropometric variables, biomarkers, and visceral fat volume. The proportion of variance in visceral fat volume explained by anthropometric measures was 65.2% in women and 60.8% in men. By using blood-based biomarkers in addition to anthropometric indices, the variance in visceral fat volume explained could be increased by 4.8% in women and 4.0% in men. After backward selection, HbA1c, triglycerides, and adiponectin remained in the final multivariable regression model in women, while in men hsCRP, leukocytes, AST (GOT), GGT, LDL, and adiponectin remained in the final model. In the present study, blood-based biomarkers moderately improved the prediction of visceral fat volume. This finding suggests that the underestimation of true associations between visceral fat and disease outcomes in epidemiological studies remains critical, even when using comprehensive sets of anthropometric and biomarker variables as proxies of visceral adiposity.

Clinical chemistry profiles in injection heroin users from Coastal Region, Kenya.

BackgroundAlthough the co-burden of injection drug use and HIV is increasing in Africa, little is known about the laboratory markers of injection drug use and anti-retroviral treatment (ART) in Kenyan injection drug users. This study, therefore, aimed at determining the clinical chemistry profiles and identifying the key laboratory markers of HIV infection during ART in injection heroin users (IHUs).MethodsClinical chemistry measurements were performed on serum samples collected from HIV-1 infected ART-experienced (n = 22), naive (n = 16) and HIV-1 negative (n = 23) IHUs, and healthy controls (n = 15) from Mombasa, coastal Kenya.ResultsHIV uninfected IHUs had lower alanine aminotransferase (ALT) levels (P = 0.023) as ART-exposed IHUs exhibited lower albumin (P = 0.014) and higher AST to platelet index (APRI) (P < 0.0001). All IHUs presented with lower aspartate aminotransferase to ALT values (P = 0.001) and higher C-reactive protein (CRP) levels (P = 0.002). ART-naive IHUs had higher globulin levels (P = 0.013) while ART-experienced and naive IHUs had higher albumin to total protein (P < 0.0001) and albumin to globulin (P < 0.0001) values. In addition, CD4+ T cells correlated with ALT (ρ = −0.522, P = 0.011) and CRP (rho, ρ = 0.529, P = 0.011) in HIV negative and ART-experienced IHUs, respectively. HIV-1 viral load correlated with albumin to globulin index in ART-experienced (ρ = −0.468, P = 0.037) and naive (ρ = −0.554, P = 0.040) IHUs; and with albumin to total protein index (ρ = −0.554, P = 0.040) and globulin (ρ = 0.570, P = 0.033) in ART-naive IHUs.ConclusionAbsolute ALT, albumin, globulin, and CRP measurements in combination with APRI, AST to ALT, albumin to total protein and albumin to globulin indices may be useful laboratory markers for screening IHUs for initiating and monitoring treatment.