Cyclic-di-GMP stimulates keratinocyte innate immune responses and attenuates methicillin-resistant Staphylococcus aureus colonization in a murine skin wound infection model.

BACKGROUND: Staphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model. RESULTS: Stimulation of HaCaT cells with 125 µM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human ß-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 µM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice. CONCLUSIONS: These results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.

Neferine prevents ultraviolet radiation-induced skin photoaging.

The aim of the present study was to evaluate the anti-photoaging effect of neferine upon exposure of mice to ultraviolet (UV) radiation. An in vivo photoaging model was established by repeatedly exposing mouse dorsal skin to UV-A and UV-B radiation for 12 weeks. Through skin photographs, hematoxylin and eosin staining, Masson's trichrome staining, and scanning and transmission electron microscopy, skin wrinkles, epidermal thickness and dermal collagen were analyzed in the UV-irradiated mouse skin. Furthermore, the levels of endogenous antioxidants, namely superoxide dismutase (SOD) and glutathione peroxidase (GPx), were measured to determine the extent of UV-induced oxidative stress that was associated with photoaging. The results demonstrated that the topical application of neferine following UV irradiation reduced oxidative stress by increasing SOD and GPx activities, and attenuated the photoaging process. Histological and ultrastructural examination revealed that neferine delayed skin wrinkle formation by inhibiting epidermal hypertrophy and collagen loss and degradation. In conclusion, the results of the present study indicated that neferine effectively prevents UV-induced skin photoaging and photodamage.

Catechol cross-linked antimicrobial peptide hydrogels prevent multidrug-resistant Acinetobacter baumannii infection in burn wounds.

Hospital-acquired infections are common in burn patients and are the major contributors of morbidity and mortality. Bacterial infections such as Staphylococcus aureus (S. aureus) and Acinetobacter baumannii (A. baumannii) are difficult to treat due to their biofilm formation and rapidly acquiring resistance to antibiotics. This work presents a newly developed hydrogel that has the potential for treating bacterial wound infections. The hydrogel formulation is based on an antimicrobial peptide (AMP), epsilon-poly-l-lysine (EPL) and catechol, which was cross-linked via mussel-inspired chemistry between the amine and phenol groups. In vitro studies showed that EPL-catechol hydrogels possess impressive antimicrobial and antibiofilm properties toward multidrug-resistant A. baumannii (MRAB). In addition, cytotoxicity study with the clonal mouse myoblast cell line (C2C12) revealed the good biocompatibility of this hydrogel. Furthermore, we created a second-degree burn wound on the mice dorsal skin surface followed by contamination with MRAB. Our results showed that the hydrogel significantly reduced the bacterial burden by more than four orders of magnitude in infected burn wounds. Additionally, there was no significant histological alteration with hydrogel application on mice skin. Based on these results, we concluded that EPL-catechol hydrogel is a promising future biomaterial to fight against multidrug-resistant bacterial infections.

Characterization of CRISPR-Cas systems in Leptospira reveals potential application of CRISPR in genotyping of Leptospira interrogans.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. However, understanding of the pathogenic mechanism of Leptospira is still elusive due to the limited number of genetic tools available for this microorganism. Currently, the reason for the genetic inaccessibility of Leptospira is still unknown. It is well known that as an acquired immunity of bacteria, Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR-associated gene (CRISPR-Cas) systems can help bacteria against invading mobile genetic elements. In this study, the occurrence and diversity of CRISPR-Cas systems in 41 genomes of Leptospira strains were investigated. Three subtypes (subtype I-B, subtype I-C and subtype I-E) of CRISPR-Cas systems were identified in both pathogenic and intermediate Leptospira species but not in saprophytic species. Noteworthy, the majority of pathogenic species harbor two different types of CRISPR-Cas systems (subtype I-B and subtype I-E). Furthermore, Cas2 protein of subtype I-C in L. interrogans exhibited a metal-dependent DNase activity in a nonspecific manner. CRISPR spacers in subtype I-B are highly conserved within the same serovars and hypervariable across different serovars of L. interrogans. Based on the subtype I-B CRISPR arrays, the serotypes of different L. interrogans strains were easily identified. Investigation of the origin of CRISPR spacers showed that 192 spacers (23.5%) matched to mobile genetic elements, indicating CRISPR-Cas systems may play an important role in the defense of foreign invading DNA.

Mussel-Inspired Hydrogel with Potent in Vivo Contact-Active Antimicrobial and Wound Healing Promoting Activities.

Open wounds (e.g., burns and trauma) are always challenged by various opportunistic bacteria. There is an urgent need for developing novel wound dressing that is able to prevent bacterial infection and promote the healing simultaneously. Herein, we developed a new type of antimicrobial hydrogels for the open wound healing through imitating a facile mussel-inspired catechol/polyamine chemistry. This hydrogel was prepared using catechol (CT) and ε-poly-l-lysine (EPL) by oxidative cross-linking directly in the open air at room temperature. This nonleaching CT/EPL hydrogel not only exhibited excellent contact-active antimicrobial activities against Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive "superbug" methicillin-resistant Staphylococcus aureus (MRSA) but also inhibited the biofilm formation in vitro. Moreover, the animal burn wound model study clearly validated the in vivo anti-infective property of CT/EPL hydrogel against MRSA infection. More importantly, the CT/EPL hydrogel possessed low cytotoxicity and enhanced cell migration in vitro. A full-thickness cutaneous wound model study revealed that CT/EPL hydrogel upregulated the expression of vascular endothelial growth factor (VEGF) and reduced the production of the pro-inflammatory cytokines, thus promoted the wound healing. These findings suggested that the CT/EPL hydrogel have great potential as a wound dressing for preventing bacterial infection and accelerating healing of open wounds.

Antioxidative and antiphotoaging activities of neferine upon UV-A irradiation in human dermal fibroblasts.

Our daily exposure to ultraviolet radiation (UVR) results in the production of reactive oxygen species (ROS), lipids, proteins and DNA damage and alteration in fibroblast structure, thus contributing to skin photoaging. For this reason, the use of natural bioactive compounds with antioxidant activity could be a strategic tool to overcome ultraviolet A (UV-A) induced deleterious effect. Neferine is an alkaloid extract from the seed embryos of lotus (Nelumbo nucifera Gaertn). In the present study, we report the protective effect of neferine against UV-A induced oxidative stress and photoaging in human dermal fibroblasts (HDFs). HDFs subjected to UV-A irradiation showed increased production of ROS and malondialdehyde (MDA). Furthermore, it depleted the cellular enzymatic antioxidant superoxide dismutase (SOD) and non-enzymatic antioxidant glutathione peroxidase (GPx). On the other hand, HDFs treated with neferine followed by UV-A irradiation reversed the process, reduced the ROS and lipid peroxidation and restored the antioxidants pool. Moreover, neferine treatment significantly inhibited UV-A induced matrix metalloproteinase-1 (MMP-1) expression in HDFs. Remarkable morphological and ultrastructural alterations observed in HDFs upon UV-A irradiation, were also reduced with neferine treatment. Taken together, our results suggest that neferine has strong antioxidative and photoprotective properties and thus may be a potential agent for the prevention and treatment of UV-A mediated skin photoaging.

Case Report: Cerebral leukodystrophy and the gonadal endocrinopathy: a rare but real association.

A 30 year old married Pakistani woman presented in January 2018 with an eight month history of progressive left sided weakness, ataxia, spasticity, underdeveloped secondary sexual characteristics and primary infertility. She was the elder sister of a 19 year old bed bound woman who was diagnosed with vanishing white matter (VWM) disease 12 months previously. The MRI scan of the brain  demonstrated diffuse leukodystrophy and her hormonal assays were significant for premature ovarian failure. Results from her genetic tests demonstrated a point mutation in eukaryotic initiation factor 2B (EIF2B). Thus, she was the second confirmed case of  VWM from her  family of 12 siblings with normal parents.

Protective effect of neferine against UV-B-mediated oxidative damage in human epidermal keratinocytes.

BACKGROUND: Studies have shown that skin exposure to ultraviolet radiation (UVR) results in the formation of reactive oxygen species (ROS), thus altering the cellular function. The human epidermal skin layer is mainly composed of keratinocytes, which is damaged by UV-B radiation-induced intracellular oxidative stress. Neferine is an alkaloid extract from lotus seed embryos and is known to promote antioxidant activity. OBJECTIVE: In this study for the first time, we investigated the photoprotective action of neferine, against UV-B-produced oxidative damage in human epidermal keratinocytes (HEKs). METHODS: We established an in Vitro study model using HEKs. Cellular viability was determined by MMT assay kits. The intracellular oxidative stress was measured using ROS and malondialdehyde (MDA) assay kits. Endogenous antioxidants were measured by superoxide dismutase (SOD) and glutathione peroxidase (GPx) assay kits. Photoprotective nature of neferine was further evaluated by analyzing the morphological and ultrastructural alterations in keratinocytes. RESULTS: Neferine inhibit the UV-B-mediated increase in ROS and MDA levels in pretreated keratinocytes. The antioxidants, SOD and GPx activities were significantly high in neferine pretreated UV-B groups. Mitochondrial and endoplasmic reticulum damage were less evident in neferine-pretreated UV-B groups as compared with the control group, which might be associated with reduced oxidative stress and lipid peroxidation. CONCLUSION: Taken together, our results suggest that neferine can prevent UV-B-induced oxidative damage and may thus be a potential agent for prevention and treatment of skin damage and photoaging.

Classic Raymond Syndrome.

Classic Raymond syndrome presents with abducens nerve palsy on the ipsilateral side with contralateral hemiparesis and facial nerve paralysis. A 60-year gentleman presented with deviation of left angle of mouth and right sided weakness. Examination showed that he had left sided abducens nerve palsy, with contralateral central facial paralysis and paresis. MRI of brain confirmed left pontine infarct. These findings were consistent with classic Raymond syndrome. Till now, only a few cases have been reported worldwide, this being the first case reported in South Asia. This case confirms that classic Raymond syndrome is different from the common type of Raymond syndrome in terms of sparing of coritcofacial fibers in the latter type.

Mean Platelet Volume (MPV) as an indicator of disease activity and severity in lupus.

Background: Systemic lupus erythematosus (SLE) is a relatively uncommon disease of young females in Pakistan. Usually, it has a relapsing-remitting course with variable severity and disease activity. Amongst the different clinical and laboratory parameters used to monitor disease activity in lupus, mean platelet volume (MPV) is a novel biomarker. Although MPV has been studied in other rheumatological conditions like rheumatoid arthritis, its role in adult SLE needs to be defined, especially in Pakistan.  Methods: The aim of this study was to evaluate the role of MPV as a biomarker of disease activity in SLE. This study included 25 patients with active SLE, and another 25 participants with stable, inactive lupus. MPV was measured in each group and compared using SPSS version 16. MPV was also correlated with SLE disease activity index (SLEDAI) and erythrocyte sedimentation rate (ESR). Independent sample t-test and Pearson's correlation tests were applied. Sensitivity and specificity of MPV were checked through ROC analysis.  Results: The MPV of patients with active SLE (n=25, mean [M]=7.12, SD=1.01) was numerically lower than those in the inactive-SLE group (n=25, M= 10.12, SD=0.97), and this was statistically significant ( P<0.001). MPV had an inverse relationship with both ESR (r=-0.93, P<0.001) and SLEDAI (r= -0.94, P<0.001). However, there was a strong positive correlation between ESR and SLEDAI (r=0.95, P<0.001). For MPV, a cutoff value of less than 8.5fl had a sensitivity of 92% and a specificity of 100% ( P< 0.001).  Conclusions: Higher disease activity in SLE is associated with a correspondingly low MPV.

Psoriatic Arthritis Is an Indicator of Significant Renal Damage in Patients with Psoriasis: An Observational and Epidemiological Study.

Background. Psoriasis affects joints in around 30% of the patients. Recent studies have demonstrated an increased risk of essential hypertension, ischemic heart disease, and stroke in psoriatic patients. However, the prevalence of renal disease in patients with psoriasis has not been evaluated properly. Objectives. Objectives were to evaluate renal functions in patients with psoriasis and to assess any possible relationship of renal failure with psoriasis and psoriatic arthritis. Methods. In this cross-sectional study, 30 participants were recruited into the following three groups: group-A, psoriatic arthritis; group-B, psoriasis without arthritis; and group-C, healthy subjects. Renal function tests were performed for every participant of each group. The data was analyzed by using SPSS version 16. Chi-squared and one-way ANOVA tests were applied, considering a P value of less than 0.05 as a standard criterion. Results. Serum creatinine, urea, and phosphate were the highest in group-A, higher in group-B, and normal in group-C, P < 0.05. Similarly, GFR was the lowest in group-A, lower in group-B, and normal in group-C. The difference in mean GFR values was statistically significant, F(2) = 355, P < 0.001. Moreover, proteinuria (gm/day) was seen in 96.7% of the patients with psoriatic arthritis, (M = 1.18 ± 0.55, P < 0.05) against 10% of the psoriatic patients without arthritis (M = 0.41 ± 0.10, P < 0.05). Conclusion. Derangement of renal function is more prevalent in psoriatic patients, especially in those with concomitant psoriatic arthritis. Therefore, each psoriatic patient must be routinely screened for an underlying renal failure.

Vanishing White Matter (VWM) Disease Presenting As Neuro-Ovarian Failure.

A 19-year girl was admitted with a one-month history of worsening spastic paraparesis, cerbellar ataxia, visual decline and worsening headaches on a background of walking difficulty, progressive quadriparesis and migraine since the age of 10 years. She had no sensory loss, and cranial nerves examination was notable for optic atrophy with crescent formation only. She had primary amenorrhea and underdeveloped secondary sexual characteristics. Ultrasonograhic studies of the pelvis confirmed small ovaries, and uterus. The magnetic resonance imaging (MRI) of the brain showed diffuse leukodystrophy. A diagnosis of leuko-ovarian syndrome or vanishing white matter (VWM) disease was made on the basis of Van der Knaap criteria. To the best of their knowledge, the authors are most probably reporting the first ever case of this rare clinical entity from Pakistan with special focus on its diagnostic and management challenges in the light of limited retrospective case reviews.

Lujan-Fryns Syndrome (LFS): A Unique Combination of Hypernasality, Marfanoid Body Habitus, and Neuropsychiatric Issues, Presenting as Acute-Onset Dysphagia.

BACKGROUND: Lujan-Fryns syndrome (LFS) is an extremely rare, X-linked disorder, for which the full clinical spectrum is still unknown. Usually, it presents with neuropsychiatric problems such as learning disabilities and behavioral issues in a typical combination with marfanoid features. Often, there is a positive family history for the disorder. However, sporadic cases have also been reported in males. More interestingly, there is no case of LFS presenting with acute-onset dysphagia in the English language medical literature. CASE PRESENTATION: A 17-year-old Pakistani mentally normal school boy was admitted for the workup of acute-onset dysphagia, hypernasal speech, and nasal regurgitation of liquids. He had no neuropsychiatric issues, and his family history was unremarkable. An obvious nasal twang, facial dysmorphism, and marfanoid body habitus were found on examination. The genetic tests revealed a pathogenic missense mutation in the MED12 gene on his X-chromosome. CONCLUSION: LFS can present as acute-onset dysphagia and in the absence of any neuropsychiatric issues or positive family history of the syndrome.

Hyperammonemia Is Associated with Increasing Severity of Both Liver Cirrhosis and Hepatic Encephalopathy.

Background. Hyperammonemia resulting from chronic liver disease (CLD) can potentially challenge and damage any organ system of the body, particularly the brain. However, there is still some controversy regarding the diagnostic or prognostic values of serum ammonia in patients with over hepatic encephalopathy, especially in the setting of acute-on-chronic or chronic liver failure. Moreover, the association of serum ammonia with worsening Child-Pugh grade of liver cirrhosis has not been studied. Objective. This study was conducted to solve the controversy regarding the association between hyperammonemia and cirrhosis, especially hepatic encephalopathy in chronically failed liver. Material and Methods. In this study, 171 cirrhotic patients had their serum ammonia measured and analyzed by SPSS version 16. Chi-squared test and one-way ANOVA were applied. Results. The study had 110 male and 61 female participants. The mean age of all the participants in years was 42.33 ± 7.60. The mean duration (years) of CLD was 10.15 ± 3.53 while the mean Child-Pugh (CP) score was 8.84 ± 3.30. Chronic viral hepatitis alone was responsible for 71.3% of the cases. Moreover, 86.5% of participants had hepatic encephalopathy (HE). The frequency of hyperammonemia was 67.3%, more frequent in males (N = 81, z-score = 2.4, and P < 0.05) than in females (N = 34, z-score = 2.4, and P < 0.05), and had a statistically significant relationship with increasing CP grade of cirrhosis (χ2(2) = 27.46, P < 0.001, Phi = 0.40, and P < 0.001). Furthermore, serum ammonia level was higher in patients with hepatic encephalopathy than in those without it; P < 0.001. Conclusion. Hyperammonemia is associated with both increasing Child-Pugh grade of liver cirrhosis and hepatic encephalopathy.

Primary Sjogren's Syndrome Presenting as Acute Interstitial Pneumonitis/Hamman-Rich Syndrome.

A previously well, 45-year-old Pakistani lady was admitted to the medical unit on-call of Khyber Teaching Hospital (KTH) Peshawar with a 5-day history of fever, productive cough with copious mucoid sputum, dyspnea, and pleuritic chest pain. She also complained of dry eyes, mouth, and vagina. Her chest X-ray showed diffuse alveolar shadowing and arterial gas analysis confirmed type 1 respiratory failure. Over the next few days, she deteriorated rapidly making an urgent transfer to the medical intensive care unit (MICU) necessary, where she was mechanically ventilated. An HRCT followed by bronchoscopic biopsies made a diagnosis of acute interstitial pneumonitis (AIP), formerly known as Hamman-Rich syndrome. She also turned out to be positive for both anti-SS-A/Ro and anti-SS-B/La antibodies along with a positive Schirmer's test and lower lip biopsy. She received intravenous steroids and supportive care. The patient had a complete recovery after approximately three weeks' stay in the hospital with lung function returning back to normal. This is most probably the first ever case of primary Sjogren syndrome (pSjS) presenting as AIP, recovering completely in less than a month time.

Coexisting giant splenic artery and portal vein aneurysms leading to non-cirrhotic portal hypertension: a case report.

BACKGROUND: Splenic artery aneurysms are the commonest visceral and third most common abdominal artery aneurysms, having a strong association with both pregnancy and multiparity. Here we report possibly the first case of a giant splenic artery aneurysm in association with a smaller portal vein aneurysm, in a woman who had never conceived, leading to non-cirrhotic portal hypertension. CASE PRESENTATION: A 40-year-old Pakistani Asian woman who had no evidence of liver cirrhosis presented in April 2016 for a diagnostic workup of ascites, massive splenomegaly, and pancytopenia. An abdominal ultrasound followed by computed tomography angiography showed a giant aneurysm in her splenic artery and another smaller one in her portal vein. She underwent splenectomy and excision of the splenic artery aneurysm. Surgical findings included a giant splenic artery aneurysm pressing on her portal vein and causing its aneurysmal dilatation. On her first review in July 2016, she was generally in good health, ascites had subsided, and her full blood count was normal. Her portal vein aneurysmal dilatation, which was presumed to be secondary to the pressure effect from the splenic artery aneurysm, had shrunken remarkably in size. CONCLUSION: A giant splenic artery aneurysm can cause non-cirrhotic portal hypertension and should be treated with splenectomy and aneurysmectomy.

Epithelioid Haemangioendothelioma (EHE) of the Liver.

A45-year previously known hypertensive lady presented with 2-year history of upper abdominal pain, heaviness, and weight loss. Her radiological assessment suggested the possibility of hepatoma or liver metastases. Considering her age, overall good health and absence of cirrhosis, a liver biopsy was taken which showed hepatic epithelioidhaemangioendothelioma (HEHE), a rare and unusual intermediate grade vascular tumor which can easily be confused with hepatoma or metastatic liver disease. To the best of their knowledge, the authors are most probably reporting the first ever case of HEHE from Pakistan with special emphasis on its clinical presentations, clinico-radiological diagnostic clues, and the management options in the light of the limited retrospective studies.

Ferritin Is a Marker of Inflammation rather than Iron Deficiency in Overweight and Obese People.

Background. In clinical practice, serum ferritin is used as a screening tool to detect iron deficiency. However, its reliability in obesity has been questioned. Objectives. To investigate the role of ferritin in overweight and obese people, either as a marker of inflammation or iron deficiency. Methods. On the basis of body mass index (BMI), 150 participants were divided into three equal groups: A: BMI 18.5-25 kg/m2, B: BMI 25-30 kg/m2, and C: BMI > 30 kg/m2. Serum iron, total iron binding capacity (TIBC), transferrin saturation, ferritin, C-reactive protein, and hemoglobin (Hb) were measured for each participant and analyzed through SPSS version 16. One-way ANOVA and Pearson's correlation tests were applied. Results. Ferritin was the highest in group C (M = 163.48 ± 2.23, P < 0.001) and the lowest in group A, (M = 152.78 ± 1.81, P < 0.001). Contrarily to ferritin, transferrin was the lowest in group C, (M = 30.65 ± 1.39, P < 0.001) and the highest in group A, (M = 38.66 ± 2.14, P < 0.001). Ferritin had a strong positive correlation with both BMI (r = 0.86, P < 0.001) and CRP (r = 0.87, P < 0.001) and strong negative correlation with Hb, iron, TIBC, and transferrin saturation (P < 0.001). Conclusion. Ferritin is a marker of inflammation rather than iron status in overweight and obese people. Complete iron profile including transferrin, rather than serum ferritin alone, can truly predict iron deficiency in such people.