Effect of platelet rich plasma versus melatonin on testicular injury induced by Busulfan in adult albino rats: a histological and immunohistochemical study.

This study was done to estimate the testicular histological alterations induced by Busulfan (BUS) and compare the possible protective effects of melatonin (MT) and platelet rich plasma (PRP) in a rat model. Sixty-four male rats were dispersed into: control group, BUS group, melatonin group, and PRP group. Blood samples were processed for biochemical analysis. Tissue specimens were managed for light and electron microscopic studies. Immunohistochemical expression of vimentin and proliferating cell nuclear antigen (PCNA) was performed. Busulfan induced severe testicular damage in all studied methodologies. It showed a statistically significant decrease in serum testosterone and elevation of MDA when compared to the control group. Abnormal testicular cytostructures suggesting defective spermatogenesis were observed: distorted seminiferous tubules, deformed spermatogenic cells, low germinal epithelium height, few mature spermatozoa, and also deformed barrier. Vimentin and PCNA expressions were reduced. Ultrastructurally, Sertoli cells and the blood testis barrier were deformed, spermatogenic cells were affected, and mature spermatozoa were few and showed abnormal structure. Both melatonin and PRP induced improvement in all the previous parameters and restoration of spermatogenesis as confirmed by improvement of Johnsen's score from 2.6 ± .74 to 7.6 ± .92. In conclusion, melatonin and PRP have equal potential to ameliorate the testicular toxicity of BUS. Melatonin can provide a better noninvasive way to combat BUS induced testicular injury.

Analysis of Toll-Like Receptor-2 and 4 Expressions in Peripheral Monocyte Subsets in Patients with Type 1 Diabetes Mellitus.

Background: Dysfunction of the peripheral blood monocytes in the form of changes in their proportion, cytokines or pattern-recognition receptors (PRR) expressions may be involved in the pathogenesis of type 1 diabetes mellitus (T1DM). Our aim is to analyze the three monocyte subsets; classical, non-classical and intermediate monocytes and their expression of Toll-like receptors 2 (TLR-2) and 4 (TLR-4) in T1DM patients. Methods: The peripheral blood monocytes of 20 T1DM patients were analyzed by Flow cytometry to measure their count and TLR-2 and TLR-4 expression. Results: T1DM patients had more non-classical and intermediate monocytes, whereas classical monocytes were comparable between patients and control (20 healthy volunteers). Classical, non-classical and intermediate monocytes had no significant correlations with hemoglobin (Hb) A1C in controls, while all subsets showed positive correlations with HbA1C in T1DM. TLR-2 and TLR-4 expression were significantly increased in classical monocytes in patients, especially those with diabetic ketoacidosis (DKA), and both of them showed positive correlations with the duration of T1DM. The expression of TLR-2 inside non-classical monocytes showed a negative correlation with LDL cholesterol and TLR-4/TLR-2 ratio showed positive correlations with the duration of T1DM and negative correlations with total cholesterol. The expression of TLR-2 inside intermediate monocytes showed positive correlations with the duration of T1DM and TLR-4/TLR-2 ratio showed negative correlations with the duration of T1DM Conclusions: The observed changes in both proportions and TLR-2 and TLR-4 expression of monocyte subsets can raise the possible role in the pathogenesis of early stages of T1DM and DKA. Abbreviations APC: allophycocyanin; CBC: complete blood picture; DKA: diabetic acidosis; DM: diabetes mellitus; FITC: fluorescein isothiocyanate; FSC: forward scatter; Hb: haemoglobin; MFI: mean channel fluorescence intensity; PE: phycoerythrin; PRR: pattern-recognition receptors; SPSS: statistical package for the social sciences; SSC: side scatter; T1DM: Type1DM; TLRs: toll-like receptors.

Hypoxia-Preconditioned Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Mitigate Hypoglycemic Testicular Injury Induced by Insulin in Rats.

Hypoglycemia is a neglected metabolic disorder. Thus, we evaluated the protective effect of hypoxia-preconditioned human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on hypoglycemic testicular injury. We examined 56 testes from 28 animals: 7 rats with insulin-induced hypoglycemia (HG group), 7 hypoglycemic rats which received an intratesticular injection of hUCB-MSCs (HG-MSC group), and 14 untreated control rats. Testosterone level, testicular catalase (CAT) activity, and malondialdehyde (MDA) level were analyzed. Immunostaining for specific testicular germ and somatic cell markers was performed. Proliferating and apoptotic cells were detected by anti-PCNA and anti-caspase-3, respectively. Morphometrical data were statistically analyzed. The hypoglycemic rats showed a significant decrease in testosterone level and CAT activity and a significant increase in MDA production. Examination of histological structure and protein expression of diverse germ cell markers revealed collapsed tubules that were lined by degenerated germ cells, decreased lactate dehydrogenase type C immune expression, as well as decreased proliferating and increased apoptotic cells number in hypoglycemic testes. Injection of MSCs improved testicular biochemical parameters, preserved germ cells and somatic cells, and decreased apoptosis. In conclusion, hypoxia-preconditioned hUCB-MSCs attenuate rat testicular injury caused by insulin-induced hypoglycemia. Avoidance and rapid management of hypoglycemia are necessary to avoid significant testicular injury.

Inhibition of Staphylococcus aureus LC554891 by Moringa oleifera Seed Extract either Singly or in Combination with Antibiotics.

Bacterial outbreaks caused by Staphylococcus aureus (S. aureus) are interesting due to the existence of multidrug resistant (MDR) isolates. Therefore, there is a need to develop novel ways to control such MDR S. aureus. In this study, some natural agents such as honey bee (HB), extracts of either Moringa oleifera seeds (MSE), or leaves (MLE) and essential oils of garlic, clove, and moringa were studied for their inhibitory activity against this S. aureus pathogen. About 100 food samples including beef luncheon (n = 25), potato chips (n = 50), and corn flakes (n = 25) were investigated for possible pollution with the S. aureus bacteria. The isolated bacteria suspected to belong S. aureus that grew well onto Baird-Parker agar (Oxoid) and shiny halo zones and positive coagulase reaction were selected and identified by API-Kits; all of them that were approved belong to S. aureus (18 strains). The sensitivity of the obtained 18 S. aureus bacterial strains to 12 antibiotics were evaluated; all of them were resistant to ofloxacin; however, other antibiotics tested showed variable results. Interestingly, the S. aureus No. B3 isolated from beef luncheon was resistant to10 antibiotics out of 12 ones tested. Multiple antibiotic resistance index (MAR) of this S. aureus strain was about 83.3%. Therefore, its identification was confirmed by sequencing of a 16S rRNA gene which approved a successful biochemical identification carried out by API Kits and such strain was designated S. aureus LC 554891. The genome of such strain appeared to contain mecA gene encoding methicillin resistance; it was found to contain hla, hlb, tsst-1, and finbA that encode α-blood hemolysis, ß-blood hemolysis, toxic shock syndrome gene, and fibrinogen-binding protein gene, respectively. In addition, the virulence factors viz. sea; seb; sec encoding enterotoxins were detected in the DNA extracted from S. aureus B3 strain. Aqueous extract of Moringa oleifera seeds (MSE) showed inhibitory activity against S. aureus LC 554891 better than that obtained by tetracycline, essential oils or HB. Minimum inhibitory concentration (MIC) of MSE was 20µg/mL. Instrumental analysis of MSE showed 14 bioactive chemical compounds. Combinations of both MSE and tetracycline showed distinctive inhibitory activity against S. aureus LC 554891 than that obtained by either tetracycline or MSE singly.

Altered Regulatory B Cell Subsets in Children with Type 1 Diabetes Mellitus.

B regulatory cells (Breg) refer to characteristic subsets of B cells that generally exert anti-inflammatory functions and maintain peripheral tolerance mainly through their ability to secrete interleukin-10 (IL10). Dysregulation in the function of Breg cells was reported in several autoimmune diseases. However, the relation between Breg and children with type 1 diabetes (T1D) is poorly understood. Thus, this study is aimed at determining whether Breg cells play a role in T1D in children or not, so we hypothesized that an altered phenotype of B cell subsets is associated with T1D in children. Children with T1D (n = 29) and control children with normal blood glucose levels (n = 14) were recruited. The percentages of different circulating IL10-producing Breg subsets, including B10, immature transitional, and plasmablasts were determined using flow cytometry analysis. Furthermore, the association between different IL10-producing B cells and patient parameters was investigated. The percentage of circulating IL10+CD24hiCD27+ (B10) and IL10+CD24hiCD38hi (immature transitional) subsets of Breg cells was significantly lower in T1D patients than in healthy controls. Moreover, these cells were also negatively correlated with fasting blood glucose and HbA1c levels. Breg cells did not correlate with autoantibody levels in the serum. These findings suggest that certain Breg subsets are numerically deficient in children with T1D. This alteration in frequency is associated with deficient islet function and glycemia. These findings suggest that Breg cells may be involved in the loss of auto-tolerance and consequent destruction of pancreatic cells and could, therefore, be a potential target for immunotherapy.

Bone marrow-derived mesenchymal stem cells ameliorate liver injury in a rat model of sepsis by activating Nrf2 signaling.

Sepsis is a fatal condition that leads to serious systemic inflammation and multiple organ dysfunction syndromes. This study was designed to investigate the possible therapeutic effect of bone marrow-derived mesenchymal stem cells (BMSCs) on sepsis-induced liver injury. We also aimed to examine the role of Nrf2 activation in modulating the response to sepsis following BMSCs treatment. Twenty-four adult male albino rats were assigned to: control, lipopolysaccharide (LPS) and LPS-stem cell groups. Liver samples were processed for light and electron microscope examinations. Immunohistochemical localization of BAX, proliferating cell nuclear antigen and nuclear factor-erythroid 2-related factor 2 (Nrf2) was carried out. Liver homogenates were prepared for assessment of reduced glutathione, glutathione peroxidase, tumor necrosis factor-alpha and interleukin-6 and also real-time PCR analysis of Nrf2 expression. BMSCs treatment improved the histopathological changes of the liver, enhanced tissue regeneration and decreased apoptosis following sepsis. We reported highly significant enhancement in Nrf2 expressions at mRNA and protein levels in the LPS-stem cell group compared with the LPS group. The up regulation of Nrf2 was probably implicated in decreasing inflammatory cytokine levels and counteracting oxidative stress induced by sepsis. Thus, BMSCs therapies could be a viable approach to treat sepsis-induced liver damage by activating Nrf2 signaling.

In neonates with vitamin D deficiency, low lymphocyte activation markers are risk factors for infection.

Background: Vitamin D has regulatory effects on different cells of the immune system and low levels are associated with several immune-mediated diseases. Aim: To investigate the association between neonatal 25-hydroxy vitamin D (25-OHD) level and the expression of lymphocyte activation markers (HLA-DR, CD69, CD25, CD45RA) on T-lymphocyte subpopulations and its impact in neonatal infection. Methods: 25-OHD level was measured in the cord blood of 56 neonates and their mothers using an enzyme immune-assay method. Based on the 25-OHD level, infants were categorised into four groups: severe deficiency (n = 7), moderate deficiency (n = 21), mild deficiency (n = 15) and normal 25-OHD level (n = 13). Mothers were classified into deficient (n = 18), insufficient (n = 21) and normal levels (n = 17). T-lymphocyte subpopulations and lymphocyte activation markers were investigated using flow cytometry. Results: There was a positive correlation between maternal and cord blood 25-OHD levels (r = 0.503, p = 0.001). The group with severe 25-OHD deficiency had the significantly lowest level of total lymphocytes, CD3+ T lymphocytes, CD4+ T-helper and CD8+ T-cytotoxic lymphocytes and CD4+CD45RA+ naïve T-cells compared with the other groups. The frequencies of CD8+CD25+, CD4+CD25+ and CD4+HLA-DR+ activated T-lymphocytes were significantly lower in the severe, moderate and mild deficiency groups than in the normal group. Seven of 43 (16.27%) infants with 25-OHD deficiency were admitted with sepsis to the neonatal intensive care unit and there were no cases of sepsis in the normal 25-OHD group. Conclusion: Vitamin D deficiency is associated with a reduction of lymphocyte subsets and altered T-lymphocyte activation which are considered to be risk factors for neonatal infection.

Functional variants in intercellular adhesion molecule-1 and toll-like receptor-4 genes are more frequent in children with febrile urinary tract infection with renal parenchymal involvement.

AIM: We studied the functional polymorphisms of intercellular adhesion molecule-1 (ICAM-1) and toll-like receptor-4 (TLR-4) genes and risk of acute pyelonephritis (APN) in children attending Assiut University Children's Hospitals, Egypt, from 2011 to 2015. METHODS: Urinary tract infections (UTIs) were diagnosed in 380 children: 98 had APN and 282 had lower UTIs. Four single-nucleotide polymorphisms in ICAM-1 and TLR-4 genes were genotyped in all subjects: ICAM-1 rs1799969 Gly241Arg, ICAM-1 rs5498 Glu469Lys, TLR-4 rs4896791 Thr399Ile and TLR-4 rs4896790 Asp299Gly. RESULTS: Patients with APN were significantly more likely to have AA genotype of the ICAM-1 rs5498 (1462 A/G) polymorphism (p = 0.04) than children with lower UTIs and the TLR-4 Asp299Gly GG genotype (p = 0.002) and G allele (p = 0.006) than healthy controls. The association with the ICAM-1 Glu469Lys (1462A/G) was less evident. The GG genotype was associated with a modest relative risk of 1.4 (p = 0.1) of developing APN, but was not an independent odds ratio, at 1.2 (p = 0.48). CONCLUSION: Functional variants in ICAM-1 and TLR-4 genes were increasingly common in children with febrile UTIs with renal parenchymal involvement, but the ICAM-1 Glu469Lys (1462A/G) association was less evident. TLR4 Asp299Gly might independently increase renal parenchymal infection rather than renal scarring.

Impact of cytokine genetic polymorphisms on the risk of renal parenchymal infection in children.

BACKGROUND: Acute pyelonephritis is associated with renal scarring in up to 30% of patients. Renal scarring may cause significant long-term morbidity. The pathogenesis of acute pyelonephritis remains unclear, although it involves interaction among uroepithelium, the immune system cells, and the locally produced cytokines. That some UTI-prone children develop acute pyelonephritis, and eventually renal parenchymal scarring, suggests a genetic role. Interleukin-6, interleukin-8, chemokine receptor-1 (CXCR1), and tumor necrosis factor-alpha (TNFα), the key regulators of the host immune responses, are proteins whose secretion is controlled by genes. We postulated that functional polymorphic variants of their genes might have a role in APN susceptibility. OBJECTIVES: We sought to investigate a possible association of the common functional polymorphisms in genes encoding IL-6, IL-8, CXCR1, and TNFα with the risk of APN in children. METHODS: Urine culture was used to diagnose 300 children with UTI, of mean age of 51.31 ± 37.4 months (2-180 months). 99Tc-DMSA scans diagnosed 86 children with APN. Follow-up scans identified new renal scars in 18 children. Six functional single-nucleotide polymorphisms (SNPs) in genes encoding IL-6, IL-8, CXCR1, and TNFα were genotyped in all subjects (IL-6 rs1800795 (-174G/C), IL-6 rs1800796 (-572G/C), IL-8 rs2227306 (781C/T), IL8 rs4073 (-251A/T), CXCR1 rs2234671 (2607G/C), and TNFα rs1800629 (-308G/A)). RESULTS: TT genotype of IL-8 -251A/T polymorphism was significantly higher in APN patients (26.7%) than those with lower UTI (11.7%, p = 0.01) and control individuals (12.2%, p = 0.002). T allele was significantly more common in APN than in lower UTI (p = 0.025) and was significantly more common in APN (46%) than in the controls (p = 0.001). Similarly, TT genotype of IL-8 781C/T polymorphism was significantly more common in APN patients (31.4%) than those with lower UTI (17.3%, p = 0.003) and the controls (14.3%, p = 0.001). T allele was significantly more common in APN (55%) than lower UTI (40%, p = 0.005) and controls (37%, p = 0.001). However, IL-8 -251A/T and +781C/T SNPs did not qualify as an independent risk for parenchymal infection (OR 1.9, 95% CI 0.68-2.6, p = 0.13 and OR 2.3, 95% CI 0.89-3.7, p = 0.091, respectively). Lower UTI did not differ from the controls. The frequency of the genotypes and alleles of IL-6, CXCR1, and TNFα SNPs did not differ significantly among the different groups of the study. CONCLUSION: IL-8 -251A/T and +781C/T SNPs are associated with susceptibility to renal parenchymal infection in children and could be implicated in APN risk. However, none of these variants could clearly and independently predict this risk.

Etiological factors of short stature in children and adolescents: experience at a tertiary care hospital in Egypt.

BACKGROUND: Accurate anthropometric measurements and critical analysis of growth data allow the clinician to promptly recognize children with short stature. The aim of this study was to determine the frequency of etiological factors causing short stature among children referred to the pediatric endocrinology clinic of Assiut University Children's Hospital, the main tertiary care center in Upper Egypt. METHODS: We conducted this descriptive observational study from May 2012 to December 2015, to analyze 637 children (boys 354, girls 283) with short stature. Evaluation included: detailed medical history, physical examination, laboratory tests, bone age and chromosomal analysis. RESULTS: Endocrinological causes accounted for 26% of short stature [of them, 11.8% had growth hormone deficiency (GHD)], 63.6% had normal variants of growth [of them, 42% had familial short stature (FSS), 15.8% had constitutional growth delay (CGD) and 5.5% a combination of both]. Interestingly, celiac disease (CD) constituted 6.6% of children with short stature in our cohort. CONCLUSIONS: Although potentially treatable causes such as GHD, hypothyroidism and CD accounted for a considerable percentage of short stature in our study, the majority of short stature in children had normal variations of growth. Growth hormone treatment in children, however, should be promptly initiated with specific clinical indications. CD is a not uncommon cause of short stature.

Long-term influence of sialoadenectomy on the liver of male albino rat.

Epidermal growth factor is an endocrine product of the submandibular gland; the liver is an important target of its action and is affected by sialoadenectomy. Thirty rats were used in this study and divided into group I (sham-operated animals), group II (sialoadenectomy after 4 weeks), and group III (sialoadenectomy after 10 weeks). Liver samples were processed for light and electron microscope examination. Sialoadenectomy induced mild-to-moderate liver damage which persists up to 10 weeks after the operation. This damage is manifested morphologically rather than functionally, affecting the general structure, hepatocytes, hepatic stellate cells, and hepatic sinusoids.

Impact of common functional polymorphisms in renin angiotensin system genes on the risk of renal parenchymal scarring following childhood urinary tract infection.

BACKGROUND: Pathogenesis of renal parenchymal scarring (RPS) after acute pyelonephritis (APN) is unclear. The risk of RPS varies markedly among individuals, suggesting a genetic role. OBJECTIVE: To investigate a possible role of common polymorphisms in renin angiotensin system genes in APN-associated RPS in children. PATIENTS AND METHODS: This study included 104 APN children and 300 controls. APN was diagnosed by urine culture and typical findings on 99Tc-DMSA scans. Voiding cystourethrogram tested the presence of vesicoureteral reflux (VUR). Follow-up DMSA scans were performed 4-6 months later to identify new RPS. Angiotensin converting enzyme gene I/D, angiotensin II receptor type-1 A1166C and angiotensinogen M235T polymorphisms were genotyped. RESULTS: New RPS developed in 44.2% (46/104) of children with APN. VUR was diagnosed in 35.6% (37/104) of APN cases. RPS developed in 73% of cases of VUR. The D allele of ACE gene I/D polymorphism was significantly more common in APN cases with RPS (73.91%) than non-RPS (58.6%) and controls (54.5%) (p = 0.021, p = 0.002, respectively). The AGTR-1 A1166C A allele was significantly more common in VUR than the non-reflux children (91.9% versus 76.1%; p = 0.005). VUR, in contrast to the D allele (OR 6.1, 95% CI 0.878-19.7; p = 0.05), was an independent risk factor for RPS. DISCUSSION: ACE gene D allele is associated with a twofold increase in RPS risk, which could be a result of a functional effect to increase tissue levels and activity of ACE during APN. However, D allele failed to qualify as an independent risk and its RPS association could be dependent on other co-factors, such as TGFß1 activation, or the D-allele might link with recently discovered functional polymorphisms at the 5' end of the ACE gene. Although VUR is an independent risk for RPS, it is not clear whether this is due to exposure of the kidneys to infected urine, or VUR-associated dysplasia. In contrast with published literature, we noted higher rates of RPS and high-grade VUR, suggesting a more aggressive VUR course or local unawareness of APN. Our study has its limitations; the small number of VUR children, and the clinical and ethical difficulties of testing VCUG and DMSA in controls. CONCLUSIONS: ACE gene D allele is associated with, but cannot independently predict, RPS in children. VUR is an independent risk for post-pyelonephritic scarring. AGTR-1 1166A/C polymorphism is associated with occurrence, but not progression, of VUR.

Growth Parameters and Vitamin D status in Children with Thalassemia Major in Upper Egypt.

AIM: The aim of this study is to assess the growth parameters, vitamin D, calcium, and phosphorous status in children with thalassemia major receiving packed red cells transfusion with chelation therapy. PATIENTS AND METHODS: In a case control study, 100 patients with beta thalassemia major (aged from 4 to 15 years) were compared with 100 sex- and age-matched children serves as a control group. Anthropometric measurement, Serum level of calcium, phosphorus and vitamin D (25 hydroxycholecalciferol) were estimated for all patients & controls. RESULTS: 49% of our patients had short stature. 47% were underweight. BMI of 43 (43%) patients were low. The mean total serum calcium (6.6±1.2 mg/dl) and 25-hydroxycholecalciferol (25-OH Vit D) (10.4±4.6 mcg/dl) levels were significantly lower in our patients than in controls (10.2±1.06 mg/dl and 40.2±12.3 mcg/dl, respectively); each P< 0.001. CONCLUSION: Children with beta thalassemia major have delayed growth and metabolic abnormalities that signify the importance of therapeutic interventions. The presence of these abnormalities may be due to iron overload and poor nutritional support.

Functional polymorphisms in transforming growth factor-beta-1 (TGFbeta-1) and vascular endothelial growth factor (VEGF) genes modify risk of renal parenchymal scarring following childhood urinary tract infection.

BACKGROUND: The risk of renal scar formation following urinary tract infection (UTI) varies markedly between individuals. We sought to investigate a possible role of the common polymorphisms in the gene encoding for VEGF and TGFbeta-1, key regulators of tissue repair, in renal scarring. METHODS: Acute pyelonephritis was diagnosed in 104 children (63 males) aged 2 months to 12 years by urine culture and 99Tc-DMSA renal scan. A follow-up isotope scan was performed 4-6 months later to identify new renal scar formation. Vesicoureteral reflux (VUR) was examined by micturating cystourethrogram. Controls comprised 300 healthy children with no evidence of renal disease. Three single-nucleotide polymorphisms (SNPs) in the TGFbeta-1 (-800 A/G, -509 C/T and 869 C/T) and four SNPs in the VEGF gene (-2578 C/A, -1154 G/A, -460 T/C and +405 G/C) were genotyped in all subjects. RESULTS: Forty-six of the 104 patients developed renal parenchymal scarring (44.2%). VUR was found in 35.6%. The -509 T allele in the TGFbeta-1 promoter was significantly more common in cases with renal scarring (51%) than in non-scarring patients (22.4%) and controls (23.6%) (both P < 0.0001). At the haplotype level, the GTC combination at -800/-509/+869 was strongly associated with renal scarring (P = 0.0002). VEGF-460 CC was more common in UTI cases with renal scarring than in non-scarring patients and controls (P = 0.03 and 0.001, respectively). Multiple logistic regression testing identified the presence of VUR (odds ratio 12.4, CI 3.8-40; P < 0.001) and the TGFbeta-1 -509 T allele (OR 6.1, CI 2.4-15.5; P < 0.001) as independent risk factors for renal scarring after UTI. In contrast, age, gender and the type of underlying organism were not predictive of renal scarring. CONCLUSIONS: Activating variants in the TGFbeta-1 and VEGF gene promoters are associated with post-UTI renal scar formation in children. The TGFbeta-1 509T allele predicts renal scarring independent of VUR.