M1 polarization induction by lead and amyloid peptides in microglial cells: Implications for neurodegeneration process.

Neurodegeneration in conditions like Alzheimer's and Parkinson's disease is influenced by genetic and environmental factors. This study explores the potential neurodegenerative effects of lead (Pb) toxicity and amyloid beta peptides (Aßp 1-40 and Aßp 25-35) by promoting M1 polarization in microglial cells. To this end, we investigated and observed that IC50 concentrations of Pb (22.8 µM) and Aßp 25-35(29.6 µM). Our results demonstrated significant Pb uptake (31.13% at 25 µM Pb) and increased intracellular ROS levels (77.1%) upon treatment with Pb in combination of both Aßp 1-40 and Aßp 25-35. Protein carbonylation significantly increased (73.12 nmol/mL) upon treatment with Pb in combination of both Aßp 1-40 and Aßp 25-35, indicating oxidative damage and compromised cellular defenses against oxidative stress along with elevated DNA oxidative damage (164.9 pg/mL of 8-OH-dG) upon treatment with Pb in combination with both Aßp 1-40 and Aßp 25-35. Microglial polarization showed elevated M1 markers (inducible nitric oxide synthase and cyclooxygenase 2) and reduced M2 markers (arginase-1 and cluster of differentiation 206), suggesting Pb's role in inducing neurodegenerative microglial polarization. These findings provide insights into the complex molecular events contributing to Pb-induced neurotoxicity and neurodegenerative diseases.

Heavy Metal Mediated Progressive Degeneration and Its Noxious Effects on Brain Microenvironment.

Heavy metals, including lead (Pb), cadmium (Cd), arsenic (As), cobalt (Co), copper (Cu), manganese (Mn), zinc (Zn), and others, have a significant impact on the development and progression of neurodegenerative diseases in the human brain. This comprehensive review aims to consolidate the recent research on the harmful effects of different metals on specific brain cells such as neurons, microglia, astrocytes, and oligodendrocytes. Understanding the potential influence of these metals in neurodegeneration is crucial for effectively combating the ongoing advancement of these diseases. Metal-induced neurodegeneration involves molecular mechanisms such as apoptosis induction, dysregulation of metabolic and signaling pathways, metal imbalance, oxidative stress, loss of synaptic transmission, pathogenic peptide aggregation, and neuroinflammation. This review provides valuable insights by compiling the supportive evidence from recent research findings. Additionally, we briefly discuss the modes of action of natural neuroprotective compounds. While this comprehensive review aims to consolidate the recent research on the harmful effects of various metals on specific brain cells, it may not cover all studies and findings related to metal-induced neurodegeneration. Studies that are done using bioinformatics tools, microRNAs, long non-coding RNAs, emerging disease models, and studies based on the modes of exposure to toxic metals are a future prospect to be explored.

Unveiling Potential Neurotoxic Mechansisms: Pb-Induced Activation of CDK5-p25 Signaling Axis in Alzheimer's Disease Development, Emphasizing CDK5 Inhibition and Formation of Toxic p25 Species.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with an etiology influenced by various genetic and environmental factors. Heavy metals, such as lead (Pb), have been implicated in AD pathogenesis, but the underlying mechanisms remain poorly understood. This study investigates the potential neurodegenerative role of Pb and amyloid ß peptides (1-40 and 25-35) via their interaction with cyclin-dependent kinase 5 (CDK5) and its activator, p25, in an attempt to unravel the molecular basis of Pb-induced neurotoxicity in neuronal cells. To this end, a CDK5 inhibitor was utilized to selectively inhibit CDK5 activity and investigate its impact on neurodegeneration. The results revealed that Pb exposure led to elevated Pb uptake (56.7% at 15 µM Pb) and disturbances in intracellular calcium (19.6% increase upon Pb treatment). The results revealed a significant decrease in total antioxidant capacity (by 88.6% upon Pb treatment) and also elevation in protein carbonylation (by 26.2% upon Pb and Aßp's combination treatment), indicative of oxidative damage, suggesting an impaired cellular defence against oxidative stress and elevated DNA oxidative damage (178 pg/ml and 182 pg/ml of 8-OH-dG upon Pb and All treatment). Additionally, dysregulations in levels of calpain, p25-35 and CDK5 are observed and markers associated with antioxidant metabolism (phospho-Peroxiredoxin 1), DNA damage responses (phospho-ATM and phospho-p53), and nuclear membrane disruption (phospho-lamin A/C) were observed, supporting the role of Pb-induced CDK5-p25 signaling in AD pathogenesis. These findings shed light on the intricate molecular events underlying Pb-induced neurotoxicity and provide valuable insights into the mechanisms that contribute to AD development.

Cowpea isoflavones enhance the osteoblast differentiation and antioxidant capacity in synergy with vitamin D and ß-carotene: A mechanistic in vitro study.

Background: Osteoporosis (OS) is a pathological condition that makes bones susceptible to fractures by affecting the balance between bone formation and resorption. Recent literature uncovered the possible potential of bioactive compounds with antioxidant mechanisms to counter the issue. Cowpea (CP) isoflavones based on our previous study, vitamin D and natural antioxidant ß-carotene for its pleotropic protective effects were assessed alone and in combination. Aim: The study aims to assess the antioxidant and osteoblast differentiation abilities of cowpea isoflavones alone and in combination of vitamin D (VD) and ß-carotene (BC) in the human osteosarcoma cell line Saos2. Methods: Saos2 cells were maintained in cell culture conditions and concentrations of CP extract (genistein + daidzein), BC and VD required to increase cell proliferation were estimated using MTT assay. Upon treating cells with the EC50 concentrations, lysates were prepared and levels of alkaline phosphatase (ALP) and osteocalcin were evaluated using ELISA. Oxidative stress parameters and osteoblast differentiation markers were evaluated. Results: CP extract (genistein + daidzein), BC and VD concentrations which enhanced the cell proliferation rate were determined and elevated levels of ALP and osteocalcin upon treatment was observed. Anti-oxidant stress parameters studied showed an increase in cells upon treatment compared to control. Significant alterations in levels of protein involved in osteoblast differentiation are observed upon treatment. Conclusion: Cowpea isoflavones has shown a significant activity against OS by elevating antioxidant parameters and inducing osteoblast differentiation in the present study.

Exposure of combination of environmental pollutant, lead (Pb) and ß-amyloid peptides causes mitochondrial dysfunction and oxidative stress in human neuronal cells.

Exposure to the environmental pollutant lead (Pb) has been linked to Alzheimer's disease (AD), in which mitochondrial dysfunction is a pathological consequence of neuronal degeneration. The toxicity of Pb in combination with ß-amyloid peptides (1-40) and (25-35) causes selective death in neuronal cells. However, the precise mechanism through which Pb induces Alzheimer's disease, particularly mitochondrial damage, is unknown. Changes in mitochondrial mass, membrane potential, mitochondrial complex activities, mitochondrial DNA and oxidative stress were examined in neuronal cells of human origin exposed to Pb and ß-amyloid peptides (1-40) and (25-35) individually and in different combinations. The results showed depolarization of mitochondrial membrane potential, decrease in mitochondrial mass, ATP levels and mtDNA copy number in Pb and ß-amyloid peptides (1-40) and (25-35) exposed cells. Also, significant reductions in the expression of mitochondrial electron transport chain (ETC) complex proteins (ATP5A, COXIV, UQCRC2, SDHB, NDUFS3), as well as down regulation of ETC complex gene expressions such as COXIV, ATP5F1 and NDUFS3 and antioxidant gene expressions like MnSOD and Gpx4 were observed in exposed cells. Furthermore, Pb and ß-amyloid peptides exposure resulted in elevated mitochondrial malondialdehyde levels and a decrease in mitochondrial GSH levels. Our findings suggest that Pb toxicity could be one of the causative factors for the mitochondrial dysfunction and oxidative stress in Alzheimer's disease progression.

Anti-cancer activity of pyridoxal phosphate and metformin combination in human pancreatic cancer cells.

Background: Pancreatic cancer is the foremost cause of cancer-related deaths in many developed countries with a poor prognosis. With advanced disease conditions chemotherapy, surgery followed by radiation is the regimen to prolong the survival. But a complete cure is questionable. Metformin is the first-line drug used for the treatment of type 2 diabetes in the world. Aim: The study aims to assess the anti-cancer activity of metformin with the combination of micronutrient pyridoxal phosphate (PLP) in the human pancreatic cancer cell line (PANC-1). Methods: Panc1 cells were maintained in vitro cell culture conditions. The IC50 concentrations of metformin and PLP were estimated and selected by using MTT assay. Morphological changes upon treatments were observed under microscope. Distribution of cells pattern was observed with propidium iodide dye in cell cycle assay. Different phases of cell distribution were studied with apoptosis assay. Results: More morphological changes were observed with PLP followed metformin. MTT assay revelled the IC50 concentrations of metformin and PLP were 20.95 ± 0.98 mM and 5.70 ± 0.07 mM. The cell cycle assay revealed that the percentage of cells was arrested in different phases with the treatments. Apoptosis assay revelled metformin increased necrosis population to 9.9%, whereas PLP has enhanced to 14.2% apoptosis. Tumour suppressor protein p53 levels had increased to 24.8% with PLP and 3.5% with metformin. Conclusion: In conclusion, PLP has significantly induced cell cycle arrest, apoptosis and enhanced p53 protein expression but a combination of PLP with metformin drug has not synergised anti-cancer activity in human PANC1 cells.

Defective mitophagy and induction of apoptosis by the depleted levels of PINK1 and Parkin in Pb and ß-amyloid peptide induced toxicity.

Exposure to lead (Pb), an environmental pollutant, is closely associated with the development of neurodegenerative disorders through oxidative stress induction and alterations in mitochondrial function. Damaged mitochondria could be one of the reasons for the progression of Alzheimer's Disease (AD). Mitophagy is vital in keeping the cell healthy. To know its role in Pb-induced AD, we investigated the PINK1/Parkin dependent pathway by studying specific mitophagy marker proteins such as PINK1 and Parkin in differentiated SH-SY5Y cells. Our data have indicated a significant reduction in the levels of PINK1 and Parkin in cells exposed to Pb and ß-amyloid peptides, both Aß (25-35) and Aß (1-40) individually and in different combinations, resulting in defective mitophagy. Also, the study unravels the status of mitochondrial permeability transition pore (MPTP), mitochondrial mass, mitochondrial membrane potential (MMP) and mitochondrial ROS production in cells treated with individual and different combination of Pb and Aß peptides. An increase in mitochondrial ROS production, enhanced MPTP opening, depolarization of membrane potential and reduced mitochondrial mass in the exposed groups were observed. Also, in the present study, we found that Pb and ß-amyloid peptides could trigger apoptosis by activating the Bak protein, which releases the cytochrome c from mitochondria through MPTP that further activates the AIF (apoptosis inducing factor) and caspase-3 proteins in the cytosol. The above findings reveal the potential role of mechanisms like PINK1/Parkin mediated mitophagy and dysfunctional mitochondria mediated apoptosis in Pb induced neurotoxicity.

Assessment of Antioxidant, Immunomodulatory Activity of Oxidised Epigallocatechin-3-Gallate (Green Tea Polyphenol) and Its Action on the Main Protease of SARS-CoV-2-An In Vitro and In Silico Approach.

Owing to the instability of Epigallocatechin Gallate (EGCG), it may undergo auto-oxidation and form oxidised products or dimers. In the present study, we aimed to evaluate the therapeutic effects, including antioxidation and immunomodulatory action, of the Oxidised Epigallocatechin Gallate (O-EGCG) as compared to native EGCG and the action of these compounds on main protease (Mpro) docking against SARS-CoV-2. HCT-116 (Human Colon Cancer) cell lines were used to estimate the total antioxidant capacity and lipid peroxidation levels and pro-inflammatory markers (human IL-6, IL-1ß, TNF-α). Further, molecular docking analysis was performed by AutoDock and visualised in Discovery studio. Improved antioxidant capacity of O-EGCG was observed, and there was a significant decrease in the inflammatory markers (IL-1ß, IL-6, and TNF-α) when O-EGCG was applied as compared to EGCG. The O-EGCG was shown to be strongly associated with the highest docking score and active site residues of IL-1, IL-6, and TNF- α, as well as the Mpro of SARS-CoV-2, according to in silico approach. The in vitro and in silico analyses indicate an improved therapeutic action of the oxidised form of EGCG. The effective inhibitory action of O-EGCG against SARS-CoV-2 suggests further exploration of the compound against COVID-19 and its efficacy. However, in vivo studies and understanding of the mechanism of action of O-EGCG may yield a better opinion on the use of O-EGCG and future human clinical trials.

Mechanisms associated with the dysregulation of mitochondrial function due to lead exposure and possible implications on the development of Alzheimer's disease.

Lead (Pb) is a multimedia contaminant with various pathophysiological consequences, including cognitive decline and neural abnormalities. Recent findings have reported an association of Pb toxicity with Alzheimer's disease (AD). Studies have revealed that mitochondrial dysfunction is a pathological characteristic of AD. According to toxicology reports, Pb promotes mitochondrial oxidative stress by lowering complex III activity in the electron transport chain, boosting reactive oxygen species formation, and reducing the cell's antioxidant defence system. Here, we review recent advances in the role of mitochondria in Pb-induced AD pathology, as well as the mechanisms associated with the mitochondrial dysfunction, such as the depolarisation of the mitochondrial membrane potential, mitochondrial permeability transition pore opening; mitochondrial biogenesis, bioenergetics and mitochondrial dynamics alterations; and mitophagy and apoptosis. We also discuss possible therapeutic options for mitochondrial-targeted neurodegenerative disease (AD).

Mitochondria-Mediated Moderation of Apoptosis by EGCG in Cytotoxic Neuronal Cells Induced by Lead (Pb) and Amyloid Peptides.

The developmental, epigenetic, and epidemiological studies on lead (Pb) toxicity have reported a strong connection between lead exposure and the progression of Alzheimer's disease (AD). The amyloid peptides were the main triggering elements, in the generation of extracellular plaques through which multiple cellular signaling events such as apoptosis and primarily oxidant-antioxidant balancing system will be affected, which leads to neuronal cell death. Our previous studies indicated that epigallocatechin gallate (EGCG), abundantly present in green tea, was found to be effective in alleviating the metal-induced neurotoxicity at the cellular level in terms of cell viability and apoptosis The aim of this study was to explore the protective mechanism of EGCG on the markers of oxidant-antioxidant system and mitochondria, which are involved in metal-induced neuronal cell death. Initially, the IC50 values for lead(Pb-5 µM), amyloid peptides (AP(1-40)-60 µM; AP(1-40)-8 µM), and EC50 value for EGCG(50 µM) were determined by both time- (12 h, 24 h, 48 h) and concentration-dependent manner and analyzed by MTT assay. The experimental groups were designed initially by treating with Pb and APs individually and in different combinations along with the presence of EGCG and are compared to the Pb and AP treated group without EGCG exposure. The cell lysates were used for analyzing oxidative stress markers by standardized laboratory protocol and the expression of mitochondrial markers such as VDAC and cytochrome C which were analyzed by both western blot and real-time PCR. Our results indicate that the EGCG-treated group has shown a significant increase in antioxidant marker expression levels (GSH, SOD, catalase, vitamin C) and a decrease in oxidative stress marker (NOS, MDA) levels when compared to the group without EGCG treatment (p < 0.05). Similarly, a significant decrease in expression levels of VDAC and cytochrome c were observed in the EGCG-treated group when compared to the group without EGCG treatment (p < 0.05). Our approach revealed that EGCG protects SH-SY5Y cells from Pb- and AP-induced cytotoxicity by regulating voltage-dependent anion channel (VDAC) expression and oxidant-antioxidant system and inhibits neuronal cell death.

Inactivation of GAP-43 due to the depletion of cellular calcium by the Pb and amyloid peptide induced toxicity: An in vitro approach.

Environmental pollutant, Lead (Pb) is known to induce neurotoxicity in human. The central nervous system is the most vulnerable to the minute levels of Pb induced toxicity. Pb has been linked to Alzheimer's disease (AD) as a probable risk factor, as it shows epigenetic and developmental link associated with Alzheimer's disease-like pathology. Beta amyloid peptides were considered as the crucial factors in the beta amyloid plaque formation in Alzheimer's disease brain. In this context, we investigated the molecular mechanism involved in the development of Pb induced Alzheimer's disease in in vitro. Previous data from our studies have reported that Pb in the presence of beta Amyloid peptide (1-40) and (25-35) induces more apoptosis than individual exposures. Here, to further evaluate the molecular mechanism underlying Pb induced Alzheimer's disease; we focussed on the involvement of calcium signalling in inducing cell death. Our experimental observations suggesting that Pb in the presence of beta amyloid peptide alters intracellular calcium levels, which leads to the increased beta-secretase activity, which further promotes the generation of beta amyloid peptides. It also showed depression in the levels of GAP-43 expression, inhibition of PKC activity and altering synaptic activity further leads to cell death.

Mitigative effects of epigallocatechin gallate in terms of diminishing apoptosis and oxidative stress generated by the combination of lead and amyloid peptides in human neuronal cells.

Environmental exposure to lead (Pb) is reported to associate with the development of Alzheimer's disease, where the formation of ß-amyloid peptides (APs) of (1-40), (1-42), and (25-35) is considered as the major risk factor. In this context, we aimed at investigating the effect of epigallocatechin gallate (EGCG), a major flavonoid polyphenol available in green tea, in mitigating the individual and combined toxicity generated by Pb and ß-APs in terms of oxidative stress and apoptosis in human neuronal cells. SH-SY5Y cells were exposed to Pb and ß-APs of (1-40) and (25-35) individually and in different combinations in the presence and absence of EGCG. The results indicated that EGCG mitigated both Pb- and ß-AP-induced oxidative stress in scavenging reactive oxygen species and apoptosis by improving the expression levels of Bax and bcl2 and inhibiting annexin V and caspase-3. Thus, our study shows that EGCG protects SH-SY5Y cells against the cytotoxicity induced by Pb and ß-APs by decreasing oxidative stress and inhibiting apoptosis.

Isoflavones rich cowpea and vitamin D induces the proliferation and differentiation of human osteoblasts via BMP-2/Smad pathway activation: Mechanistic approach.

Isoflavones, such as Genistein (Ge) and Daidzein (Dz) are widely studied Phytoestrogens with potent anti-osteoporotic and good antioxidant activity. Cowpea is one such legume having high isoflavone content and hence we aimed at studying the beneficial effects of the isoflavones isolated from cowpea as it is widely accepted staple food in India. Previously, we reported the effect of Cowpea isoflavones (CP) and Vitamin D (VD) owing to its ability of improving the osteoporotic condition in a diet induced osteoporotic rat model. In the present study, we tried to explore the underlying mechanism of CP and VD along with positive controls Dz and Ge in influencing the functions of human osteoblasts at cellular level. Initially, MG-63 cells were assessed for the expression of genes involved in BMP-2 signaling pathway, like Bone morphogenic protein (BMP-2), transcription factor Osterix (OSX), total and phosphorylated Smad 1/5/8 levels and osteoblast specific genes levels namely Alkaline phosphatase (ALP), Osteopontin (OPN), and Collagen by immunoblot, flow cytometry, and quantitative RT-PCR studies. All the levels that were upregulated with the initial exposure of the compounds got inhibited after Noggin exposure a specific BMP-2 antagonist both at protein level and m-RNA level, except OSX where the expression was totally hindered in CP and Ge treated groups alone. Hence, CP and VD activate BMP-2/Smad signaling pathway and promote further proliferation and differentiation of osteoblasts. Therefore, results prove that isoflavones isolated from cowpea could be used in treating bone-related disorders.

Predictors of severe tricuspid regurgitation in patients with permanent pacemaker or automatic implantable cardioverter-defibrillator leads.

Patients with permanent pacemaker or automatic implantable cardioverter-defibrillator (AICD) leads have an increased prevalence of tricuspid regurgitation. However, the roles of cardiac rhythm and lead-placement duration in the development of severe tricuspid regurgitation are unclear. We reviewed echocardiographic data on 26 consecutive patients who had severe tricuspid regurgitation after permanent pacemaker or AICD placement; before treatment, they had no organic tricuspid valve disease, pulmonary hypertension, left ventricular dysfunction, or severe tricuspid regurgitation. We compared the results to those of 26 control subjects who had these same devices but no more than mild tricuspid regurgitation. The patients and control subjects were similar in age (mean, 81 ±6 vs 81 ±8 yr; P = 0.83), sex (male, 42% vs 46%; P = 0.78), and left ventricular ejection fraction (0.60 ±0.06 vs 0.58 ± 0.05; P = 0.4). The patients had a higher prevalence of atrial fibrillation (92% vs 65%; P=0.01) and longer median duration of pacemaker or AICD lead placement (49.5 vs 5 mo; P < 0.001). After adjusting for age, sex, and right ventricular systolic pressure by multivariate logistic regression analysis, we found that atrial fibrillation (odds ratio=6.4; P = 0.03) and duration of lead placement (odds ratio=1.5/yr; P = 0.001) were independently associated with severe tricuspid regurgitation. Out study shows that atrial fibrillation and longer durations of lead placement might increase the risk of severe tricuspid regurgitation in patients with permanent pacemakers or AICDs.

Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors.

Betulinic acid is a natural product possessing abundant and favourable biological activity, including anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties, while causing minimal toxicity to unaffected cells. The full biological potency of betulinic acid cannot be fully unlocked, however, for a number of reasons, a primary one being its limited solubility in aqueous and biologically pertinent organic media. Aiming to improve the water solubility of betulinic acid without disrupting its structurally related bioactivity, we have prepared different ionic derivatives of betulinic acid. Inhibition bioassays on HIV-1 protease-catalysed peptide hydrolysis indicate significantly improved performance resulting from converting the betulinic acid to organic salt form. Indeed, for one particular cholinium-based derivative, its water solubility is improved more than 100 times and the half maximal inhibitory concentration (IC(50)) value (22 µg mL(-1)) was one-third that of wide-type betulinic acid (60 µg mL(-1)). These encouraging results advise that additional studies of ionic betulinic acid derivatives as a therapeutic solution against HIV-1 infection are warranted.

Predictors for the development of severe tricuspid regurgitation with anatomically normal valve in patients with atrial fibrillation.

BACKGROUND AND AIMS: Atrial fibrillation (AF) may be a risk factor for severe functional tricuspid valve regurgitation (FTR). We aimed to determine the predictors of severe FTR in patients with AF. METHODS AND RESULTS: From our echocardiographic laboratory database, we searched for and reviewed the medical records of consecutive patients with severe FTR and AF seen at Mayo Clinic in Arizona from 2002 through 2009. Our search identified 42 patients who met all inclusion criteria. These patients (cases) with severe FTR and AF were compared with 38 patients (controls) with AF who had no greater than mild tricuspid regurgitation. Case patients with severe FTR were older than controls (mean, 81 years vs. 76 years; P < 0.001) and more frequently had chronic AF (69% vs 26%; P < 0.001). Mean right atrial volume (86 mL/m(2) vs 46 mL/m(2) ; P < 0.001), right ventricular volume (42 mL ± 33 mL vs 22 mL ±8 mL; P < 0.001) and tricuspid annular diameter (3.6 cm vs 3.0 cm; P < 0.001) were larger in cases than in controls. Patients with severe FTR also had a higher prevalence of right-sided heart failure (69% vs 16%; P < 0.001). After adjusting for age and gender, right atrial and right ventricular volumes were independent predictors for the development of severe FTR in patients with AF (odds ratio, 1.7 [95% CI, 1.3-2.8] for every 10 mL/m(2) increase in right atrial volume; P = 0.0002 and odds ratio, 3.1 [95% CI, 1.5-8.9] for every 10 mL increase in right ventricular volume; P = 0.0002). CONCLUSIONS: Severe FTR occurs in older patients with chronic AF as a result of marked right atrial and right ventricular dilatation; and enlargement of the tricuspid annulus in the absence of pulmonary hypertension. More importantly, severe FTR leads to increased prevalence of right-sided heart failure underscoring the nonbenign nature of chronic AF.

Takotsubo cardiomyopathy in a patient with pulmonary embolism.

Takotsubo cardiomyopathy is an acute cardiac syndrome characterized by transient regional wall motion abnormalities of the left ventricular apex or midventricle. Patients often present with chest pain or dyspnoea, ST-segment elevation, and minor elevations of cardiac enzyme levels. Takotsubo cardiomyopathy has been associated with severe emotional or physical stress such as severe burns, spinal cord injury, subarachnoid haemorrhage, and multiple traumas. We report a case of takotsubo cardiomyopathy in a 79-year-old woman with pulmonary embolism. Although pulmonary embolism has been listed as a potential cause of takotsubo cardiomyopathy, this is the first case reported with this association.