Molecular Characterization and Mutational Analysis of Clarithromycin- and Levofloxacin-Resistance Genes in Helicobacter pylori from Gastric Biopsies in Southern Croatia.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic-protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

Low-dose mannitol (0.3 g kg(-1)) improves the pulsatility index and minimum diastolic blood flow velocity in traumatic brain injury.

OBJECTIVE: The aim of the study was to investigate the effects of using low-dose mannitol (0.3 g kg(-1)) on the pulsatility index (PI) and minimum diastolic blood flow velocity (FV-min) of the middle cerebral artery in a traumatic brain injury (TBI). METHODS: Low-dose mannitol (0.3 g kg(-1)) was administered to a group of 20 patients with a TBI. Transcranial Doppler (TCD) ultrasonography was used to monitor the PI and FV-min. The study included patients with a diffuse traumatic brain injury and Glasgow coma score < 8. The initial TCD ultrasonography values were pathological (PI > 1.4 and FV-min < 20 cm s(-1)). TCD ultrasonography examinations were carried out before mannitol administration, immediately after administration and 1, 2 and 3 hours after the administration of mannitol. RESULTS: A one-way analysis of variance revealed significant changes in the PI (F = 8.392; p < 0.001) and FV-min (F = 8.291; p = 0.001) after the use of mannitol. CONCLUSIONS: Low-dose mannitol administration appears to be efficacious for improving the indicators of disturbed circulation in a TBI (FV-min increase, PI decrease). The maximum decrease in the PI was recorded 1 hour after the administration of mannitol and was 10.9% of the initial value. The maximum increase in the FV-min was recorded 1 hour after administration and was 29.7% of the initial value. These changes were significant ∼ 2 hours later.

Epidermal growth factor receptor (EGFR) expression in patients with laryngeal squamous cell carcinoma.

To assess the epidermal growth factor receptor (EGFR) correlation with histopathologic and clinical characteristics of laryngeal squamous cell carcinoma (SCC) and the impact of EGFR overexpression on patient survival. This retrospective study included 185 SCC patients treated at Clinical Department of ENT, Head and Neck Surgery, Split University Hospital Center between January 1, 2000 and December 31, 2009. A statistically significant correlation (p < 0.001) was recorded between the level of EGFR expression and SCC histopathologic grade, stage, metastasizing potential, relapsing potential, and patient survival. Kaplan-Meier survival curve yielded a statistically significant difference (χ(2) = 75.05; p < 0.001) among the four patient groups with different levels of EGFR expression. The higher the level of EGFR expression, the poorer is the patient prognosis and survival. In our study, expression of EGFR as a biomarker showed a potential predictive value in laryngeal SCC.

Depression and serum interleukin-6 levels in patients on dialysis.

BACKGROUND: Depression is a common psychiatric problem in patients undergoing dialysis. Several studies have been performed to validate the association between depression and inflammation in haemodialysis patients. The levels of proinflammatory cytokines are increased in chronic renal failure patients, as in depression. The objective of this study was to compare the incidence of depression in the patients on dialysis (on hemodialysis /HD/ and on continuous ambulatory peritoneal dialysis /CAPD/), and a relationship between depression and the presence of inflammation. SUBJECTS AND METHODS: 88 patients (52 on HD and 36 on CAPD) were enrolled in this study. Depressive symptoms were measured with the Beck Depression Inventory (BDI). The BDI is a 21-item self-report instrument, and the elevated symptoms of depression were defined as a BDI score ≥16. HD patients were treated with high-flux polysulphone biocompatible dialyzers and CAPD patients were treated with usual dwell time (4-6 hours during the day and 8-10 hours at night). The presence of an inflammatory state was assesded by determinations of plasma interleukin-6 (IL-6) levels. RESULTS: Depression (BDI ≥16) was present in 28.4% of dialysis patients, 35% of patients on hemodialysis (HD) and 18.1% of patients on continous ambulatory peritoneal dialysis (CAPD). The BDI score was significantly lower in CAPD patients comparing to HD patients, as well as the levels of albumin, C-reactive protein (CRP) and interleukin-6 (IL-6). IL-6 serum levels were similar in patients with depression and patients without depression in the whole group, as in HD patients. In CAPD patients without depression IL-6 levels were significantly lower. CONCLUSIONS: The prevalence of depression was higher in HD comparing to CAPD patients. Although IL-6 level was higher in HD compared to CAPD patients, the relationship between depression and presence of inflammation parametars were observed in CAPD, but not in HD patients.

Enthesitis in IBD Patients.

Inflammatory bowel disease (IBD) is marked by chronic inflammation of the gastrointestinal tract and encompasses two major subtypes, Crohn's disease (CD) and ulcerative colitis (UC). IBD is frequently accompanied by extraintestinal manifestations (EIMs), with axial and peripheral spondyloarthritis (SpA) being the most common. Enthesitis, an inflammation of the bone insertions of capsules, ligaments, and tendons, represents an initial lesion in SpA. However, enthesitis remains an underestimated and often obscured EIM. The early detection of subclinical entheseal involvement in IBD patients using ultrasound (US) could provide an opportunity for timely intervention. US is a more feasible and affordable approach than magnetic resonance imaging (MRI). While previous meta-analyses have reported on the incidence and prevalence of SpA in IBD, specific attention to enthesitis has been lacking. Therefore, this narrative review aims to assess the current knowledge on existing IBD-SpA cohorts, focusing specifically on enthesitis.

Growth differentiation factor-15 serum concentrations reflect disease severity and anemia in patients with inflammatory bowel disease.

BACKGROUND: Population of patients with inflammatory bowel disease (IBD) is burdened by various extraintestinal manifestations which substantially contribute to greater morbidity and mortality. Growth-differentiation factor-15 (GDF-15) is often over-expressed under stress conditions, such as inflammation, malignancies, heart failure, myocardial ischemia, and many others. AIM: To explore the association between GDF-15 and IBD as serum concentrations of GDF-15 were shown to be an independent predictor of poor outcomes in multiple diseases. An additional aim was to determine possible associations between GDF-15 and multiple clinical, anthropometric and laboratory parameters in patients with IBD. METHODS: This cross-sectional study included 90 adult patients diagnosed with IBD, encompassing both Crohn's disease (CD) and ulcerative colitis (UC), and 67 healthy age- and sex-matched controls. All patients underwent an extensive workup, including colonoscopy with subsequent histopathological analysis. Disease activity was assessed by two independent gastroenterology consultants specialized in IBD, employing well-established clinical and endoscopic scoring systems. GDF-15 serum concentrations were determined following an overnight fasting, using electrochemiluminescence immunoassay. RESULTS: In patients with IBD, serum GDF-15 concentrations were significantly higher in comparison to the healthy controls [800 (512-1154) pg/mL vs 412 (407-424) pg/mL, P < 0.001], whereas no difference in GDF-15 was found between patients with CD and UC [807 (554-1451) pg/mL vs 790 (509-956) pg/mL, P = 0.324]. Moreover, multiple linear regression analysis showed that GDF-15 levels predict CD and UC severity independent of age, sex, and C-reactive protein levels (P = 0.016 and P = 0.049, respectively). Finally, an association between GDF-15 and indices of anemia was established. Specifically, negative correlations were found between GDF-15 and serum iron levels (r = -0.248, P = 0.021), as well as GDF-15 and hemoglobin (r = -0.351, P = 0.021). Accordingly, in comparison to IBD patients with normal hemoglobin levels, GDF-15 serum levels were higher in patients with anemia (1256 (502-2100) pg/mL vs 444 (412-795) pg/mL, P < 0.001). CONCLUSION: For the first time, we demonstrated that serum concentrations of GDF-15 are elevated in patients with IBD in comparison to healthy controls, and the results imply that GDF-15 might be involved in IBD pathophysiology. Yet, it remains elusive whether GDF-15 could serve as a prognostic indicator in these patients.

Prognostic value of MVD, LVD and vascular invasion in lymph node-negative colon cancer.

BACKGROUND/AIMS: To clarify the influence of microvessel density (MVD), lymphangiogenesis (LVD), and vascular invasion on prognosis in lymph node-negative colon cancer. METHODOLOGY: We performed immunohistochemical analysis from 152 Duke's B colon carcinomas, CD34 and LYVE-1 antibodies. Carcinomas were graded as low or high grade. χ2 test was used to examine their relationships and correlations with clinicopathological parameters. Survival time was analyzed and the differences between groups were assessed. RESULTS: A statistically significantly correlation was found between increasing MVD with age >60 years, tumor size >4cm, and poor tumor differentiation (χ2=40.018, p<0.001). The increase in MVD was associated with shorter DFS (p<0.001) and shorter OS in patients with colon cancer (p<0.001). LVD was statistically significantly associated with increasing the number of newly created blood vessels (χ2=96.6, p<0.001), low degree of tumor differentiation (χ2=96.6, p<0.001), and vascular invasion (χ2=51.8, p<0.001) in colon cancer. Log rank analysis showed that positive staining for MVD and LVD, high histological grade, vascular invasion, male gender, and age >60 years were connected with shorter survival of patients with Dukes B colon cancer 45 vs. 100 months (p=0.016 to <0.001). CONCLUSIONS: Positive expression MVD and LVD was significantly correlation with survival time and with high tumor grade and vascular invasion in patients with Dukes B colon cancer. The correlation of MVD and LVD with vascular invasion in Dukes B colon cancer indicates the need for further confirmation as a possible predictive marker.

Expression Pattern of DAB Adaptor Protein 2 in Left- and Right-Side Colorectal Carcinoma.

Left-sided and right-sided colorectal cancer (L-CRC and R-CRC) have relatively different clinical pictures and pathophysiological backgrounds. The aim of this study was to investigate the presence of DAB adapter protein 2 (DAB2) as a potential molecular mechanism that contributes to this diversity in terms of malignancy and responses to therapy. The expression of the suppressor gene DAB2 in colon cancer has already been analyzed, but its significance has not been fully elucidated. Archived samples from 34 patients who underwent colon cancer surgery were included in this study, with 13 patients with low-grade CRC and 21 with high-grade CRC. Twenty of the tumors were R-CRC, while 14 were L-CRC. DAB2 expression was analyzed immunohistochemically in the tumor tissue and the colon resection margin was used as a control. Tumors were divided into L-CRC and R-CRC, with splenic flexure as the cutoff point for each side. The results showed that R-CRC had lower DAB2 protein expression compared to L-CRC (p = 0.01). High-grade tumors had reduced DAB2 expression compared to low-grade tumors (p = 0.02). These results are consistent with the analysis of DAB2 gene expression data that we exported from the TCGA Colon and Rectal Cancer Study (COADREAD). In 736 samples of colon cancer, lower DAB2 gene expression was found in R-CRC compared to L-CRC (p < 0.0001). DAB2 gene expression was significantly higher in the sigmoid colon than in the cecum and ascending colon (p < 0.01). The analysis confirmed a lower expression of the DAB2 in tumors with positive microsatellite instability (p < 0.001). In conclusion, DAB2 has a role in the biological differences between R-CRC and L-CRC and its therapeutic and diagnostic potential needs to be further examined.

Personalized Approach in Eradication of Helicobacter pylori Infection.

The increase in antibiotic resistance to Helicobacter pylori (H. pylori) is associated with a decrease in the effectiveness of eradication therapy. Although some success has been achieved by adjusting therapeutic regimens according to local data on resistance to certain antibiotics, a new approach is needed to ensure a better therapeutic response. Tailored therapy, based on sensitivity tests to antibiotics, is increasingly proving to be a superior therapeutic option, even as a first-line therapy. Moreover, the recently published Maastricht VI guidelines emphasize utilizing a susceptibility-guided strategy in respect to antibiotic stewardship as the first choice for eradication therapy. In addition, polymerase chain reaction (PCR) technology is becoming a standard tool in the diagnosis of H. pylori infections through non-invasive testing, which further optimizes the eradication process. We provide a review regarding the current position of the individualized approach in eradication therapy and its future prospects. Based on novel understandings, the personalized approach is an effective strategy to increase the successful eradication of H. pylori infections.

Acute Liver Failure after Treatment with Rivaroxaban for Aortic Thrombosis Associated with COVID-19 Infection and Methylenetetrahydrofolate Reductase Gene Polymorphism (C677T).

Coronavirus disease-2019 (COVID-19) has become associated with prothrombotic state that could lead to severe arterial thrombotic complications. In the case of severe COVID-19 infection, hepatic dysfunction has been observed in more than 50% of patients. In this article, we present a case of aortic thrombosis associated with COVID-19 infection and methylenetetrahydrofolate reductase gene polymorphism (C677T) treated with rivaroxaban resulting in acute liver failure with fatal outcome.

Cyclooxygenase-2 in Dukes B colon cancer.

BACKGROUND/AIMS: To demonstrate immunohistochemical expression of COX-2 protein in Dukes B colon cancer and to establish a correlation with clinicopathological parameters such as: age, gender, gradus, presence of vascular invasion and patient's overall survival. METHODOLOGY: We performed immunohistochemical analysis of formalin-fixed, paraffin embedded specimens form 152 Dukes B colon carcinomas, using the COX-2 monoclonal antibody. Immunohistochemical results were scored semi-quantitatively. Carcinomas were graded as low or high grade. Survival time was analyzed by Kaplan-Meier method, and the log-rank test was used to assess the differences between groups. For multivariate analysis, Cox proportional hazard regression model was used to examine several parameters simultaneously. RESULTS: Univariate analysis showed that positive staining for COX-2, high histological grade; vascular invasion; male gender and age over 60 years, were connected with shorter survival of patients with Dukes B colon cancer (0.023< p<0.001). However, multivariate analysis have shown that the high COX-2 expression in Duke's B colon cancer was unrelated to overall patient survival (RR=1.4; p=0.311). CONCLUSION: Expression of COX-2 in tumor epithelial cells does not seem to be related to survival in patients with colon cancer Dukes B.

Randomized Clinical Trial Comparing Concomitant and Tailored Therapy for Eradication of Helicobacter pylori Infection.

As high clarithromycin resistance (>20%) in the Split-Dalmatia region of Croatia hinders the treatment of H. pylori infection, the primary objective of this study was to compare concomitant quadruple with the tailored, personalized therapy as first-line eradication treatment of H. pylori. In an open-label, randomized clinical trial, 80 patients with H. pylori infection were randomly assigned to either concomitant (esomeprazole 40 mg, amoxicillin 1 gr, metronidazole 500 mg, clarithromycin 500 mg, twice daily for 14 days) or tailored therapy in accordance with the results of the antimicrobial susceptibility testing. Eradication status was assessed 4 weeks after treatment. Eradication rates were significantly higher in tailored group than in concomitant group both in intention-to-treat (70 vs. 92.5%, p = 0.010) and per-protocol (87.5 vs. 100%, p = 0.030) analysis in the setting of increasing antibiotic resistance (clarithromycin 37.5%, metronidazole 17.5%, dual resistance 10%). Adverse effects were more frequent in the concomitant group (32.5 vs. 7.5%, p = 0.006). Tailored therapy achieves higher eradication with a lower adverse events rate. With the increasing resistance of H. pylori strains to antibiotic treatment, eradication regimes with such characteristics should be strongly considered as a reasonable choice for first-line treatment.

The influence of gastroesophageal reflux in the lung: a case-control study.

BACKGROUND AND OBJECTIVE: Many researchers have investigated the pH of exhaled breath condensate but direct measurement of pH in the lung has not been performed in vivo in humans. We hypothesized that the pH measured directly in the lung would differ between healthy subjects and patients with gastroesophageal reflux disease (GERD). We also wished to determine whether an acidic environment in the lung influences pulmonary function and DL(CO), and whether microaspiration of gastric contents directly influences non-specific inflammation in the lung. METHODS: The patients were otherwise healthy individuals who had been newly diagnosed with GERD. The control subjects were mostly volunteers who underwent bronchoscopy for different reasons. For all subjects (n = 63) a medical history was taken, and physical examination, oesophagogastroduodenoscopy, fibre-optic bronchoscopy and pulmonary function testing were performed. RESULTS: In patients with GERD the average pH in the lung was 5.13 +/- 0.43; this was significantly lower than the pH in the lung of controls 6.08 +/- 0.39 (P = 0.001). Patients with GERD had lower FEV(1)% (P = 0.035), PEF (P = 0.001), FEF(50%) (P = 0.002) and FEF(25%) (P = 0.003), while the differences in FVC% and FEF(75%) were not significant. DL(CO) (P = 0.003), as well as transfer coefficient of the lung (P = 0.001), was lower in patients with GERD. LDH levels in bronchoalveolar aspirate were higher in the patients with GERD (P = 0.001). CONCLUSIONS: This study found evidence of cell and tissue injury in the lung, a lowering of pH and higher bronchoalveolar aspirate LDH levels in patients with GERD compared with healthy subjects. These findings suggest that pulmonary function, and especially DL(CO), should be evaluated in patients presenting with GERD.

Apoptosis as a Prognostic Factor in Colorectal Carcinoma: Comparison of TUNEL Method and Immunohistochemical Expression of Caspase-3.

The development of colorectal cancer is known to be characterized by a sequence of events during which normal colonic epithelium gradually transforms to carcinoma, the adenoma-carcinoma sequence. Apoptosis plays an important role in the development and maintenance of tissue homeostasis. Currently, there is no agreement in the literature about the prognosis of apoptosis in colorectal cancer. The number of studies examining the expression of caspases in colorectal cancer is very limited, and they have not examined any correlation between expression and patient survival. This study included histologic samples from 179 patients diagnosed with colon cancer. We used the TdT-mediated X-dUTP nick end labeling method and caspase-3 labeling to identify the degree of apoptosis. Our results show that lower apoptotic index measured by TdT-mediated X-dUTP nick end labeling method and lower immnuhistochemical expression of caspase-3 is associated with shorter disease-free survival and overall survival. However, only apoptotic index is proven to be an independent survival indicator. The results of our study are consistent with the proposed models of carcinogenesis of colorectal cancer that emphasize resistance to apoptosis as a decisive factor in the progression of the disease and resistance to treatment.

Immunohistochemically detected high expression of matrix metalloproteinase-2 as predictor of poor prognosis in Duke's B colon cancer.

AIM: To demonstrate immunohistochemical expression of matrix metalloproteinase-2 (MMP-2) protein in Duke's B colon cancer and determine its correlation with age, sex, grade, presence of vascular invasion, and patients' overall survival. METHOD: The study took place from January 1995 to December 1997. We determined the expression of MMP-2 in 152 formalin-fixed, paraffin embedded specimens of Duke's B colon carcinomas by immunohistochemical analysis using MMP-2 monoclonal antibody. Immunohistochemical expression was scored semiquantitatively. Carcinomas were graded as low or high grade. Survival time was analyzed with Kaplan-Meier method, and the log-rank test was used to assess the differences between groups. Cox proportional hazard regression model was used for multivariate survival analysis. RESULT: Univariate analysis showed that positive staining for MMP-2, high histological grade, vascular invasion, male sex, and age>60 years were associated with shorter survival in patients with Duke's B colon cancer (P range from 0.023 to <0.001). Multivariate analysis showed that only MMP-2 overexpression (P<0.001; hazard ratio [HR]=3.64) and vascular invasion (P<0.001; HR=4.27) were associated with shorter overall survival. CONCLUSION: Expression of MMP-2 is an important independent indicator of shorter survival in patients with Duke's B colon cancer and should be taken into consideration in decision-making on the use of adjuvant systemic therapy in patients with Duke's B colon cancer.

Fatal colchicine poisoning by accidental ingestion of meadow saffron-case report.

A 62-year-old male died of colchicine poisoning after accidental ingestion of Colchicum autumnale (meadow saffron). He ate a salad of plant with green leaves regarded as wild garlic (Allium ursinum). A few hours later he developed symptoms of gastroenteritis and was admitted to hospital. In spite of gastric lavage, activated charcoal and supportive measures, multi-organ system failure developed over the next two days. Laboratory analysis showed highly elevated blood concentrations of hepatic enzymes, creatine kinase, lactate dehydrogenase and blood urea nitrogen, as well as leukocytopenia and thrombocytopenia. Mechanical ventilation, dopamine, noradrenaline, crystalloid solutions and fresh frozen plasma were applied but despite treatment the patient died five days after the ingestion. Post-mortem examination revealed hepatic centrilobular necrosis, nephrotoxic acute tubular necrosis, petechial bleeding in fatty tissue, blunt and shortened intestinal villi and cerebral toxic edema. Botanical identification of incriminated plant gave Colchicum autumnale which confirmed colchicine poisoning. Although the accidental ingestion of Colchicum autumnale is rare and to our knowledge only five such cases have been described in detail, this is the second fatal case in Croatia described in the last 3 years.