YAP knockdown repressed autophagy in fibroblasts to accelerate wound healing through regulating En1/mTOR axis.

OBJECTIVE: Wound repair dysfunction is becoming a major public health issue worldwide. Yes-associated protein (YAP) has previously been reported to be closely related to wound healing, while how YAP accelerates wound healing via regulating autophagy needs to be further probed. MATERIALS AND METHODS: ICR male mice were involved in two independent animal experiments; the mice were randomly allocated into control, autophagy inhibitor (3-MA) (injection), and 3-MA (drip) group or control, si-NC, si-YAP group (8 mice for each). Full-thickness excisional wounds (8 mm) in mice were created by punch to construct an in vivo wound model to observe the effects of autophagy inhibitor (3-MA) (by injection and drip) and si-YAP by electrotransfection. RESULTS: Firstly, we found that the autophagy inhibitor (3-MA) accelerated wound closure in vivo. Loss-of-function experiments subsequently revealed that YAP knockdown led to increased proliferation and migration of fibroblasts as well as reduced autophagy, resulting in accelerated wound healing. In addition, our results revealed that YAP could positively regulate Engrailed-1 (En1) expression in fibroblasts. En1 knockdown also promoted the proliferation and migration of fibroblasts, meanwhile resulting in increased mammalian target of rapamycin (mTOR) levels and reduced autophagy in fibroblasts. CONCLUSIONS: YAP knockdown repressed autophagy in fibroblasts to accelerate wound closure by regulating the En1/mTOR axis.

Multicenter prospective trial of total gastrectomy versus proximal gastrectomy for upper third cT1 gastric cancer.

BACKGROUND: The appropriate surgical procedure for patients with upper third early gastric cancer is controversial. We compared total gastrectomy (TG) with proximal gastrectomy (PG) in this patient population. METHODS: A multicenter, non-randomized trial was conducted, with patients treated with PG or TG. We compared short- and long-term outcomes between these procedures. RESULTS: Between 2009 and 2014, we enrolled 254 patients from 22 institutions; data from 252 were included in the analysis. These 252 patients were assigned to either the PG (n = 159) or TG (n = 93) group. Percentage of body weight loss (%BWL) at 1 year after surgery, i.e., the primary endpoint, in the PG group was significantly less than that of the TG group (- 12.8% versus - 16.9%; p = 0.0001). For short-term outcomes, operation time was significantly shorter for PG than TG (252 min versus 303 min; p < 0.0001), but there were no group-dependent differences in blood loss and postoperative complications. For long-term outcomes, incidence of reflux esophagitis in the PG group was significantly higher than that of the TG group (14.5% versus 5.4%; p = 0.02), while there were no differences in the incidence of anastomotic stenosis between the two (5.7% versus 5.4%; p = 0.92). Overall patient survival rates were similar between the two groups (3-year survival rates: 96% versus 92% in the PG and TG groups, respectively; p = 0.49). CONCLUSIONS: Patients who underwent PG were better able to control weight loss without worsening the prognosis, relative to those in the TG group. Optimization of a reconstruction method to reduce reflux in PG patients will be important.

CNS distribution, signalling properties and central effects of G-protein coupled receptor 4.

Information on the distribution and biology of the G-protein coupled receptor 4 (GPR4) in the brain is limited. It is currently thought that GPR4 couples to Gs proteins and may mediate central respiratory sensitivity to CO2. Using a knock-in mouse model, abundant GPR4 expression was detected in the cerebrovascular endothelium and neurones of dorsal raphe, retro-trapezoidal nucleus locus coeruleus and lateral septum. A similar distribution was confirmed using RNAscope in situ hybridisation. In HEK293 cells, overexpressing GPR4, it was highly constitutively active at neutral pH with little further increase in cAMP towards acidic pH. The GPR4 antagonist NE 52-QQ57 effectively blocked GPR4-mediated cAMP accumulation (IC50 26.8 nM in HEK293 cells). In HUVEC which natively express GPR4, physiological acidification (pH 7.4-7.0) resulted in a cAMP increase by ∼55% which was completely prevented by 1 µM NE 52-QQ57. The main extracellular organic acid, l-lactic acid (LL; 1-10 mM), suppressed pH dependent activation of GPR4 in HEK293 and HUVEC cells, suggesting allosteric negative modulation. In unanaesthetised mice and rats, NE 52-QQ57 (20 mg kg-1) reduced ventilatory response to 5 and 10% CO2. In anaesthetised rats, systemic administration of NE 52-QQ57 (up to 20 mg kg-1) had no effect on hemodynamics, cerebral blood flow and blood oxygen level dependent responses. Central administration of NE 52-QQ57 (1 mM) in vagotomised anaesthetised rats did not affect CO2-induced respiratory responses. Our results indicate that GPR4 is expressed by multiple neuronal populations and endothelium and that its pH sensitivity is affected by level of expression and LL. NE 52-QQ57 blunts hypercapnic response to CO2 but this effect is absent under anaesthesia, possibly due to the inhibitory effect of LL on GPR4.

Investigating the pressure-reducing effect of wound dressings.

OBJECTIVE: Currently, polyurethane foam dressings are commercially available from many manufacturers. However, the pressure-reducing effect is expected to differ by the formulation and combination of the main and secondary ingredients and by manufacturing method. In this study, we investigated the effects of pressure reduction using dressing materials with various structural characteristics, including polyurethane foam dressings based on the engineering point of view, focusing on the dry state. METHOD: Pressure was measured in a model that simulated compression on the sacral region in a decubitus position. Pressure was measured for different dressings: ten products, consisting of five types of material (polyurethane foam, hydropolymeric, Hydrofiber, hydrocolloid, and low-adherent absorbent). RESULTS: All dressings used in this study showed significantly reduced pressure. ALLEVYN Non-Adhesive had the lowest pressure at 35.833 ± 1.155 mmHg, and DuoDERM Extra Thin CGF had the highest pressure at 66.867 ± 1.060 mmHg. The pressure of the control was 74.667 ± 1.405 mmHg. The other dressings were: ALLEVYN Adhesive: 44.233 ± 0.777 mmHg; ALLEVYN Gentle Border: 46.967 ± 1.537mmHg; Mepilex Border: 53.867 ± 0.231 mmHg; Biatain Silicone: 56.000 ± 0.520 mmHg; TIELLE: 57.267 ± 3.403 mmHg;Versiva XC: 65.900 ± 0.800 mmHg; DuoDERM CGF: 57.267 ± 1.007 mmHg; and Melolin: 53.433 ± 1.973 mmHg. CONCLUSION: The pressure-reducing effect of dressing differs not only by material type but also by product. That is, the pressure-reducing effect can differ even if the dressings are of the same material type, such as polyurethane foam. Our study investigated only the effect of materials and structural characteristics on the cushion of dressings in the dry state. Therefore, further investigation is needed to confirm the effect of pressure reduction by dressing to meet the conditions in the clinic.

Distribution of involved abdominal lymph nodes is correlated with the distance from the esophagogastric junction to the distal end of the tumor in Siewert type II tumors.

BACKGROUND: The optimal surgical approach for Siewert type II adenocarcinoma of the esophagogastric junction (AEG) has not yet been agreed. Here we investigated whether the distance from the esophagogastric junction (EGJ) to the distal end of the tumor was related to the distribution of involved abdominal lymph nodes in Siewert type II tumors. METHODS: A total of 288 patients with pT2-4 AEG Siewert II, treated by R0 surgical resection at 7 institutions in Japan, were retrospectively investigated. The distribution of involved abdominal nodes was correlated with the distance from the EGJ to the distal end of the tumor. RESULTS: In patients where the distance from the EGJ to the distal end of the tumor was ≤30 mm, the frequency of nodal involvement along the greater curvature or antrum was low (2.2%). In contrast, in patients where the distance was >50 mm, the incidence of this nodal involvement was 20.0%. In patients where the distance was 30-50 mm incidence was intermediate (8.0%). Multivariate analyses showed that the distance from the EGJ to the distal end of the tumor was significantly related to lymph node involvement along the greater curvature or antrum (odds ratio 3.7, 95% confidence interval 1.3-11, p = 0.006). CONCLUSIONS: When the distance from the EGJ to the distal end of the tumor is ≤ 30 mm for Siewert II AEG, esophagectomy or proximal gastrectomy is sufficient from the point of view of abdominal lymphadenectomy. However, a total gastrectomy should be considered for abdominal lymphadenectomy when this distance is > 50 mm.

Inferolateral marginal orbitectomy: a simple adjuvant technique for orbital decompression.

The number of patients with mild exophthalmos, without severe eye symptoms, who wish to undergo aesthetic orbital decompression, is increasing. Removal of the lateral and inferior orbital walls is a common procedure for mild to moderate exophthalmos. However, the limited space between the globe and the orbital wall is often troublesome for surgeons introducing surgical devices. As a result, the decompression tends to be insufficient in the posterior region of the orbit. We describe a simple adjuvant surgical technique to address this limitation. Through a laterally extended, transconjunctival approach, the inferior and lateral margins of the orbit are removed in a crescent shape before the actual decompression. This manoeuvre widens the working space and offers better visibility, enabling sufficient removal of the orbital walls. The technique presented facilitates the approach to the posterior regions of the orbit, enabling surgeons to more easily perform orbital decompression.

Dose-saving isolation procedure in percutaneous ethanol sclerotherapy for venous malformations.

OBJECTIVES: To evaluate the feasibility and effectiveness of an isolation technique during ethanol injection sclerotherapy for venous malformations (VMs) in the head and neck region. METHODS: The subjects were 23 patients with 35 VM lesions in the head and neck, treated between 1999 and 2012. The mean lesion area was 3.75 ± 3.09 cm(2) (±standard deviation). We confirmed the contour of the lesions to be treated on a fully filled image on direct injection cisternography, and observed patterns of communicating drainage to systemic veins. The cisterns were evacuated by squeezing and were isolated by manual compression of the communicators. Ethanol (94.5%) with a contrast agent was then injected into both isolable and unisolable lesions, up to a total volume of 1 mL/cm(2), avoiding complications. We investigated the relationship between lesion size and injected ethanol dose, and also dose per unit area. RESULTS: Both manual evacuation by compression and isolation were performed in 20 (57.1%) isolable lesions, but not in 15 unisolable lesions. The mean injected ethanol dose was 0.65 ± 0.31 mL/cm(2) overall, 0.70 ± 0.32 in isolable and 0.59 ± 0.30 in unisolable lesions (NS). However, the injected ethanol dose was significantly lower for lesions sized >6 cm. Complete to near-complete shrinkage was observed in all isolable lesions, and in 60% of unisolable lesions (P < 0.05). Clinical outcome seemed unrelated to the injected ethanol dose or the dose per unit area. There was one case of recurrence and one complication in the unisolable lesions. No further relapses or complications were observed during the follow-up period of 38.6 ± 12.3 months. CONCLUSIONS: Clinical outcome was related to the isolability not to the injected dose. The isolation appears useful for improving the safety and effectiveness of ethanol sclerotherapy for VM.

Construction of novel immune-related signature for prediction of pathological complete response to neoadjuvant chemotherapy in human breast cancer.

BACKGROUND: The aim of this study was to construct a novel prediction model for the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) using immune-related gene expression data. PATIENTS AND METHODS: DNA microarray data were used to perform a gene expression analysis of tumor samples obtained before NAC from 117 primary breast cancer patients. The samples were randomly divided into the training (n = 58) and the internal validation (n = 59) sets that were used to construct the prediction model for pCR. The model was further validated using an external validation set consisting of 901 patients treated with NAC from six public datasets. RESULTS: The training set was used to construct an immune-related 23-gene signature for NAC (IRSN-23) that is capable of classifying the patients as either genomically predicted responders (Gp-R) or non-responders (Gp-NR). IRSN-23 was first validated using an internal validation set, and the results showed that the pCR rate for Gp-R was significantly higher than that obtained for Gp-NR (38 versus 0%, P = 1.04E-04). The model was then tested using an external validation set, and this analysis showed that the pCR rate for Gp-R was also significantly higher (40 versus 11%, P = 4.98E-23). IRSN-23 predicted pCR regardless of the intrinsic subtypes (PAM50) and chemotherapeutic regimens, and a multivariate analysis showed that IRSN-23 was the most important predictor of pCR (odds ratio = 4.6; 95% confidence interval = 2.7-7.7; P = 8.25E-09). CONCLUSION: The novel prediction model (IRSN-23) constructed with immune-related genes can predict pCR independently of the intrinsic subtypes and chemotherapeutic regimens.

Inhibitory effect of statins on inflammatory cytokine production from human bronchial epithelial cells.

Statins are 3-hydroxy-3-methylglutaryl-co-enzyme A reductase inhibitors of cholesterol biosynthesis, and have been reported to exert pleiotropic effects on cellular signalling and cellular functions involved in inflammation. Recent reports have demonstrated that previous statin therapy reduced the risk of pneumonia or increased survival in patients with community-acquired pneumonia. However, the precise mechanisms responsible for these effects are unclear. In the present study, we examined the effects of statins on cytokine production from lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Interleukin (IL)-6 and IL-8 mRNA expression and protein secretion in LPS-stimulated cells were inhibited significantly by the lipophilic statin pitavastatin and the hydrophilic statin pravastatin. As these inhibitory effects of statin were negated by adding mevalonate, the anti-inflammatory effects of statins appear to be exerted via the mevalonic cascade. In addition, the activation levels of Ras homologue gene family A (RhoA) in BEAS-2B cells cultured with pitavastatin were significantly lower than those without the statin. These results suggest that statins have anti-inflammatory effects by reducing cytokine production through inhibition of the mevalonic cascade followed by RhoA activation in the lung.

Secondary solid tumors after allogeneic hematopoietic SCT in Japan.

To evaluate the incidence and risk factors for secondary solid tumors in Japan after allogeneic hematopoietic SCT (allo-HSCT), 2062 patients who had received allo-HSCT between 1984 and 2005 were retrospectively analyzed. Twenty-eight patients who developed 30 solid tumors were identified a median of 5.6 years after transplantation. The risk for developing tumors was 2.16-fold higher than that of the age- and sex-adjusted general population. The cumulative incidence of solid tumors at 10 years after allo-HSCT was 2.4%. The risk was significantly higher for tumors of the skin, oral cavity and esophagus (standard incidental ratio 40.23, 35.25 and 10.73, respectively). No increase in gastric, colon or lung cancer, despite being the most prevalent neoplasm in the Japanese, was observed. In multivariate analysis, occurrence of chronic GVHD and malignant lymphoma as a primary disease was associated with a higher risk for developing solid tumors. Eighteen patients are still alive, and their 5-year probability of survival since diagnosis of solid tumors is 59.7%. Our data suggest that the incidence and risk factors of secondary solid tumors in Japanese allo-HSCT recipients are comparable to those reported in Western countries and emphasize that the early detection of solid tumors has a crucial role in improving OS.

Prevention of gastroduodenal content reflux and delayed gastric emptying after esophagectomy: gastric tube reconstruction with duodenal diversion plus Roux-en-Y anastomosis.

Reflux of gastroduodenal contents and delayed gastric emptying are the most common and serious problems after esophagectomy with gastric reconstruction. However, attempts to reduce the above symptoms, surgically as well as non-surgically, had no or limited effect. To address this issue, we performed retrosternal gastric reconstruction with duodenal diversion plus Roux-en-Y anastomosis (RY) in eight patients with thoracic esophageal cancer and compared the outcomes with control patients who underwent standard reconstruction. The procedure is simple, safe, and not associated with any postoperative complications. The pancreatic amylase concentrations in the gastric juice samples on postoperative day 2 were slightly lower in the non-RY group than in the RY group (1884 ± 2152 vs. 25,790 ± 23,542IU/mL, respectively, P= 0.07). Postoperative endoscopic examination showed neither reflux esophagitis nor residual gastric content in the RY group. Quality of life assessed by the Dysfunction After Upper Gastrointestinal Surgery-32 questionnaire postoperatively was significantly better in the RY group than in the non-RY group for 'decreased physical activity,''symptoms of reflux,''nausea and vomiting,' and 'pain.' The results of this pilot study suggest that gastric reconstruction with duodenal diversion plus RY seems effective in improving both the reflux and delayed gastric emptying. The benefits of this procedure need to be further assessed in a large-scale, randomized controlled trial.

The significance of abdominal para-aortic lymph node metastasis in patients with lower thoracic esophageal cancer.

Para-aortic lymph node (PALN) recurrence is often seen in patients with lower thoracic esophageal cancer treated by esophagectomy with extended lymph node dissection. However, the clinicopathological characteristics of patients with PALN metastasis and the significance of PALN dissection are unknown. A total of 283 patients with lower thoracic esophageal cancer underwent esophagectomy with lymphadenectomy at our hospital between April 1984 and March 2007. Among these 283 patients, 60 patients were enrolled in this retrospective study according to following criteria: (i) clinical T2 to T4 tumor, (ii) no clinical PALN metastasis, and (iii) received PALN dissection. PALN dissection was indicated by a tumor depth of at least T2 and no severe complications. The clinicopathological data, recurrence pattern, and overall survival were compared between patients with PALN and without PALN metastasis. The mean length of surgery was 587 min and the mean blood loss was 1383 mL. The morbidity was 33.3% and mortality was 5% in this series. Sixteen patients (26.7%) had PALN metastasis; these showed significantly more lymph node metastases (15.8 ± 13.2 vs. 3.0 ± 3.2, P < 0.0001) and significantly worse survival rates (53.3% vs. 79.9% at 1 year, 6.7% vs. 62.0% at 3 years, P < 0.0001) than patients without PALN metastasis. The incidence of lymph node recurrence (P < 0.0001) and hematogenous recurrence (P= 0.0487) was also higher in patients with PALN metastasis than in patients without PALN metastasis. Among the 16 patients with PALN metastasis, a univariate analysis revealed total number of metastatic nodes < 8 (P= 0.0325) to be a significant prognostic factor. A multivariate logistic regression analysis of the regional lymph nodes identified the invasion of the lower mediastinal nodes (hazard ratio = 6.120) and retroperitoneal nodes (hazard ratio = 15.167) to be significantly correlated with PALN metastasis. PALN metastasis is suggested to be related to the systemic spread of lymphatic metastasis even in lower thoracic esophageal cancer. PALN dissection for pathological PALN(+) patients should not be performed. It remains to be determined in future prospective studies whether patients without pathological PALN metastasis, but showing PALN micrometastasis, could achieve improved survival with PALN dissection.

70-Gene classifier for differentiation between paclitaxel- and docetaxel-sensitive breast cancers.

Association of estrogen receptor (ER), progesterone receptor (PR), HER2, Ki67 and 70-gene classifier (70-GC) with a response to paclitaxel (PAC) (n=79) or docetaxel (DOC) (n=55) was investigated in the neoadjuvant setting for breast cancer patients. Sensitivity of breast tumors to PAC, but not to DOC, was found to be significantly associated with ER negativity (P=0.003), PR negativity (P=0.007), and Ki67 positivity (P=0.007). Breast tumors classified into the responders by 70-GC showed a significantly (P=0.005) higher reduction rate to PAC and interestingly a significantly (P=0.009) lower reduction rate to DOC than those classified into the non-responders by 70-GC, suggesting that 70-GC might be useful for the differentiation of PAC-sensitive and DOC-sensitive breast tumors.

Potential pitfalls in volume reduction for the treatment of Proteus syndrome.

Proteus syndrome is a rare congenital hamartomatous condition that is characterised by a wide range of malformations. Skin and skeletal developmental malformations are common and may manifest as significant physical anomalies. However, few reports have discussed surgical treatment. A 37-year-old woman presented with disproportionate enlargement of the right buttock, which required volume reduction. The overgrown tissue was combined with vascular malformations, which in our patient were extremely thick. The patient experienced massive blood loss during the procedure, due to which a blood transfusion was performed. Pathological findings revealed partial lipohyperplasia with venous malformations. Thus, during operations including incomplete excision, massive bleeding is always a possibility, but is not commonly described in these instances.

Cortactin, an actin binding protein, regulates GLUT4 translocation via actin filament remodeling.

Insulin regulates glucose uptake into fat and skeletal muscle cells by modulating the translocation of GLUT4 between the cell surface and interior. We investigated a role for cortactin, a cortical actin binding protein, in the actin filament organization and translocation of GLUT4 in Chinese hamster ovary (CHO-GLUT4myc) and L6-GLUT4myc myotube cells. Overexpression of wild-type cortactin enhanced insulin-stimulated GLUT4myc translocation but did not alter actin fiber formation. Conversely, cortactin mutants lacking the Src homology 3 (SH3) domain inhibited insulin-stimulated formation of actin stress fibers and GLUT4 translocation similar to the actin depolymerizing agent cytochalasin D. Wortmannin, genistein, and a PP1 analog completely blocked insulin-induced Akt phosphorylation, formation of actin stress fibers, and GLUT4 translocation indicating the involvement of both PI3-K/Akt and the Src family of kinases. The effect of these inhibitors was even more pronounced in the presence of overexpressed cortactin suggesting that the same pathways are involved. Knockdown of cortactin by siRNA did not inhibit insulin-induced Akt phosphorylation but completely inhibited actin stress fiber formation and glucose uptake. These results suggest that the actin binding protein cortactin is required for actin stress fiber formation in muscle cells and that this process is absolutely required for translocation of GLUT4-containing vesicles to the plasma membrane.

The unilocular fat-cell graft.

Autologous fat grafting has become a common procedure for augmenting soft tissue. However, there are still some problems with resorption and cyst formation after injection, which mainly arise from insufficient nourishment of the transplanted fat tissues. In this study, using a mouse model, we enzymatically digested fat tissues into unilocular fat cells, and then transplanted the unilocular fat cells by sub-dermal injection to allow the transplanted cells to easily spread within the injected area. Fat tissue was harvested from a green fluorescent protein transgenic mouse (C57BL/6), and the optimal digestion time was determined to be 30 min. The fat cells were then injected into the sub-dermal layer of the head skin of a C57BL/6 mouse. As a control, minced fat without digestion was also injected. The animals were sacrificed immediately after injection and on days 1, 2 and 3, as well as at weeks 1, 2 and 4 after injection, and the recipient skins were collected for microscopic observation. The unilocular fat cells were observed to spread in a solitary manner among the recipient tissues, and no necrotic areas or cysts were observed. The minced-fat-graft control showed central necrosis in the transplanted region. In addition, the minced fat tissue needed to be injected with an 18-gauge syringe, but the unilocular fat cells could be injected with a 26-gauge syringe. Thus, the unilocular fat-cell graft was determined to be a superior alternative to conventional fat grafts.

Relationship of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers to caries status and colonization of mutans streptococci in plaque in their 2.5-year-old children.

OBJECTIVES: The aim of this study was to assess the relationships of quantitative salivary levels of Streptococcus mutans and S. sobrinus in mothers with the colonization of mutans streptococci (MS) in plaque and caries status in their 2.5-year-old children. Furthermore, the dynamics of caries status in the children was evaluated in a 2-year follow-up survey. METHODS: After oral examination of 54 mother-and-child pairs, the saliva samples from the mothers and the plaque samples from the children were collected. The levels (log DNA copies/ml saliva) of S. mutans and S. sobrinus were quantified using real-time polymerase chain reaction (PCR) assays, while MS in the plaque samples were detected using a cultivation method. In addition, 50 of the 54 children participated in a 2-year follow-up survey of caries prevalence. RESULTS: In the 2.5-year-old children, the percentage of dft-positive subjects and mean number of dft were significantly higher in the MS(+) group when compared with the MS(-) group. Findings from the 2-year follow-up survey indicated that MS(+) subjects had a persistently higher mean number of dft at 4.5 years. The 2.5-year-old children were divided into three groups based on the quantitative levels of salivary S. mutans and S. sobrinus in their mothers: those whose mothers had low levels of S. mutans (<4 log DNA copies/ml) and S. sobrinus (<2) (group 1); those whose mothers had a high level of S. mutans (> or = 4) and low level of S. sobrinus (<2) (group 2); and those whose mothers had high levels of both (> or = 4 and > or = 2, respectively) (group 3). Among the three groups, the percentages of MS(+) and dft-positive children were highest in group 3 and lowest in group 1. Furthermore, multiple logistic regression analyses revealed that grouping the mothers based on salivary level of S. mutans and S. sobrinus was an efficient means to predict both MS colonization (OR = 2.96) and prevalence of dental caries (OR = 9.39) in children at 2.5 years of age. CONCLUSIONS: In the 54 mother-and-child pairs tested, the maternal salivary levels of S. mutans and S. sobrinus determined by real-time PCR were significantly related to MS colonization in plaque as well as dental caries in their children at 2.5 years of age. Thus, determination of maternal levels of both organisms using the present cut-off values is proposed as an efficient method to indicate the risks of maternal transmission of MS and childhood dental caries.