d-Glucose and amino acid deficiency inhibits casein synthesis through JAK2/STAT5 and AMPK/mTOR signaling pathways in mammary epithelial cells of dairy cows.

Amino acids and energy deficiency lead to lower milk protein content in dairy cows. However, the known mechanisms involved in this process do not adequately explain the variability of milk protein concentration in the mammary gland. We hypothesized that a deficiency in d-glucose (d-Glc) or AA would inhibit casein synthesis by regulating signaling pathways in mammary epithelial cells. Cow mammary epithelial cells (CMEC) were subjected to combinations of 1 of 3 concentrations of d-Glc (0, 2.50, or 17.5 mM) and 1 of 3 concentrations of AA (0, 1.03, or 7.20 mM). The effect of each mixture on cell cycle stage was assessed by flow cytometry. The expression levels of ß-casein and κ-casein (encoded by CSN2 and CSN3) were measured by quantitative real-time PCR and Western blotting. Phosphorylation of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5a (Stat5a), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eukaryotic factor 4E-binding protein 1 (4EBP1) were analyzed by Western blotting. The percentages of cells in the DNA postsynthetic (G2) and DNA synthesis (S) phases would decrease, with the level of d-Glc or AA declining individually, but no interaction was observed between the d-Glc and AA effects. The CSN2 and CSN3 mRNA and protein were downregulated when d-Glc or AA decreased individually from 17.5 to 2.50 mM or from 7.20 to 1.03 mM, but d-Glc deficiency had a greater effect according to the regression analysis. The phosphorylation ratio of Jak2 (Tyr1007/1008), Stat5a (Tyr694), mTOR (Ser2448), S6K1 (Thr389), and 4EBP1 (Thr37) was downregulated with the level of d-Glc or AA decline, whereas the phosphorylation ratio of AMPK (Thr183/172) was upregulated. And the change of d-Glc level had a more marked effect than AA in regulating the activity of these signaling protein above according to the regression analysis. Thus, d-Glc or AA deficiency likely reduced casein transcription via inhibition of the Jak2/Stat5 pathway, and reduced translation via suppression of the mTOR pathway by activation of AMPK, but d-Glc deficiency had a more marked effect. These indicated that deficiency of AA, and especially Glc, suppressed proliferation of CMEC and casein gene and protein expression, associated with inhibition of JAK2/STAT5 and AMPK/mTOR signaling pathways.

[Evaluation of biocompatibility of Ti-6Al-4V scaffolds fabricated by electron beam melting].

Objective: To investigate the biocompatibility of Ti-6Al-4V scaffolds fabricated by electron beam melting(EBM). Methods: Bone marrow mesenchymal stem cells(BMSC) co-cultured with Ti-6Al-4V specimens fabricated with EBM was prepared as experimental group and the regular cells culture was employed as control. The biocompatibility was detected using CCK-8 and cytoskeleton staining. The osteogenic differentiation ability was assessed using mineralization nodule formation. A 24 mm defect was created on the right mandibular body in 12 beagles. The mandibular defects were repaired with Ti-6Al-4V scaffolds mesh fabricated by EBM. General observation, CT and histology examination was carried out to evaluated the biocompatibility of Ti-6Al-4V scaffolds in vivo. Results: CCK-8 result showed the A values of the two groups had no significant difference(P >0.05). There was no significant difference between the two groups (P>0.05). Cytoskeletal staining showed that cells were fully stretched out and grew well on T-i6Al-4V specimen. The actin fibers were arranged in parallel and stained uniformly with fluorescent. After osteogenic culture, the quantity of the nodule formation of the experimental group and control group were 5.7±0.7 and 5.1 ± 0.6, respectively(P>0.05). All animals had tolerated the surgery and healed well. CT examination showed that Ti-6Al-4V scaffolds mesh had good retention with surrounding bone and the continuity of mandible was restored. Histological examination showed that no inflammation reaction or toxity was caused in the soft tissue surrounding the scaffolds and in the liver and kidney after implantation. Ti-6Al-4V scaffolds had good retention with surrounding bone. Conclusions: Ti-6Al-4V fabricated with electron beam melting has good biocompatibility.

Use of parenteral testosterone prior to hypospadias surgery.

Surgical correction of genital defects was formerly proposed when the size of the penis was sufficient to permit easy surgical repair. To enlarge penile size, temporary stimulation with testosterone or dihydrotestosterone cream has been used; however, the results were not only inconsistent, but absorption was also variable. We report our experience with parenteral testosterone as an adjunct to reconstructive genital surgery in 25 patients aged 6-18 months from July 1999 to December 2000, including 8 with penile hypospadias, 15 with penoscrotal hypospadias, and 2 with perineal hypospadias. Each had a penis that was significantly smaller than usual. Testosterone enanthate 25 mg was given i.m. once per month for a total of three doses before surgical repair. Penile length and glans circumference were measured before therapy and at operation. Side effects such as the development of pubic hair and acne were monitored. Bone age was checked 1 year later. An increase in penile length (from 19.8 +/- 2.4 mm to 23.8 +/- 2.0 mm) and glans circumference (from 27.4 +/- 1.4 mm to 37.84 +/- 2.6 mm) was apparent in all except 2 patients (P < 0.001 for both, paired t-test). Four patients had a significant increase in either penile length or glans circumference after the initial dose so that no further injections were required. No definite secondary effects were found. Preoperative parenteral testosterone therapy thus causes a significant increase in penile length and glans circumference without apparent side effects. We suggest that this therapy prior to microphallic hypospadias repair is appropriate.

Intussusception and intestinal malrotation in an infant: a case report.

An abnormal cecum position is usually found in patients with intestinal malrotation. We report one case with intussusception and intestinal malrotation in a 10-month-old infant. An unusual radiologic imaging feature and also abnormal intussusception mass location are discussed.

Perforation of toxic megacolon in non-typhoid Salmonella enterocolitis spares young infants and is immune-mediated.

Intestinal perforation, a life-threatening complication of toxic megacolon (TM) following non-typhoid Salmonella infection, is relatively uncommon in infants less than 1 year of age. The situation, also found in typhoid fever, appears to be cytokine-mediated. This finding may justify immunotherapy for older children with TM associated with non-typhoid Salmonella infection in order to prevent this complication.

Antiretroviral resistance mutations among pregnant human immunodeficiency virus type 1-infected women and their newborns in the United States: vertical transmission and clades.

To assess the impact of antiretroviral resistance on perinatal transmission prevention efforts, human immunodeficiency virus type 1 (HIV-1) genotypic resistance testing was done for 220 HIV-1-infected, zidovudine (AZT)-exposed pregnant women and 24 of their infected infants. The women were prospectively enrolled in 4 US cities in 1991-1997. Phylogenetic and sequencing analyses revealed 5 women with non-clade B infections traced to western African origins. AZT-associated mutations were detected in 17.3% of pregnant women, whereas genotypic resistance to nonnucleoside reverse-transcriptase inhibitors and protease inhibitors was infrequent. No significant association was detected between perinatal transmission and the presence of either AZT or nucleoside reverse-transcriptase inhibitor resistance-associated mutations. AZT resistance mutations were detected in 2 (8.3%) neonatal samples, but the mutation pattern was not identical to the mother's. Although no effect of viral resistance on mother-infant transmission was demonstrated, the advent of more-potent drug classes and the potential for the rapid emergence of resistance warrant prospective surveillance.

Effect of N(G)-nitro-L-arginine methyl ester on intestinal permeability following intestinal ischemia-reperfusion injury in a rat model.

Subclinical intestinal ischemia-reperfusion injury (IRI) causes an increase in mucosal permeability and may represent an early event in the pathogenesis of necrotizing enterocolitis in premature infants. Previous studies suggested that continuous, endogenous formation of nitric oxide (NO) maintains the mucosal integrity of the intestine, thus protecting the gut from injuries from blood-borne toxins and tissue-destructive mediators. This study was undertaken to assess whether the inhibition of NO production causes an increase in intestinal permeability in rats following IRI. Sprague-Dawley rats weighing 200-300 g were divided into 4 groups: (1) untreated group (normal control); (2) ischemia-reperfusion group; (3) early N(G)-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of NO production, treatment group, and (4) late L-NAME treatment group. Transient IRI was induced by 30-min occlusion, followed by reperfusion of the isolated ileal loop. The L-NAME was administered 15 min before and after mesenteric ischemia as a 25-mg/kg bolus. Fluorescein isothiocyanate-dextran (FITC-D) was used to quantitatively assess the alteration in mucosal permeability of the intestine. There was no significant increase in the portal vein FITC-D level among normal controls, ischemia-reperfusion group and late L-NAME-treated group, but there was an approximately 6-fold increase in the early L-NAME treatment group. The pathological features of the intestine following IRI include denudation of the villus epithelium and reduction of villus height, associated with marked inflammatory cell infiltration over the lamina propria. These results suggest that endogenous NO may play a role in the protecting intestinal integrity after IRI.

Subtyping HIV-1 infections in Taiwan using peptide-enzyme immunoassay, reverse transcription-polymerase chain reaction, and sequencing.

BACKGROUND AND PURPOSE: There are important questions about epidemiologic transmission patterns as well as the possibility that genetic and phenotypic differences in human immunodeficiency virus type 1 (HIV-1) affect transmissibility, infectivity, pathogenicity, and response to therapy and vaccines. To delinate the genetic heterogeneity of HIV-1 and the association of subtypes with risk factors and location of residence in Taiwan, subtypes of HIV-1 in Taiwanese patients were identified and a phylogenetic study was performed. In addition, the accuracy of peptide-enzyme immunoassay (EIA) using serum samples from Taiwanese patients infected with HIV-1 was investigated. METHODS: Peptide-EIA was used to give a preliminary subtype of HIV-1-positive serum samples collected from different areas of Taiwan. Reverse transcription (RT)-polymerase chain reaction (PCR) and genetic sequencing were used to confirm the peptide-EIA results and to construct a phylogenetic tree. RESULTS: Among the 149 serum samples, 98 were subtype B (66%), 38 subtype E (25%), two subtype Thai-B (1.3%), one subtype G (0.7%), and one subtype C (0.7%). Comparison of risk factors for HIV-1 infection and subtype revealed that most B subtype infections (59/98) occurred in homosexual or heterosexual patients, whereas 28 of 38 E subtype infections occurred in heterosexual patients. The B/E ratio was significantly different (p < 0.05) in Taipei than in other areas of Taiwan. CONCLUSIONS: These results suggest that the predominant subtype of HIV-1 infection in Taiwan is B, followed by E, and that the distribution of HIV-1 subtypes in Taiwan is similar to that of Thailand, although the genetic sequences are distinct. Homosexuality, heterosexuality, bisexuality, and intravenous drug use behaviors affect the distribution of different subtypes of HIV-1 infection. Peptide-EIA in conjunction with RT-PCR and sequencing can provide accurate subtyping of HIV-1 infection.

Septic arthritis of the hip caused by Salmonella typhi.

We describe septic arthritis of the hip in a child with typhoid fever. The aetiological diagnosis was confirmed by a positive Widal test as well as by isolation of Salmonella typhi from joint aspirate. Treatment with ceftriaxone along with surgical drainage was successful.

Sonographic evaluation of the pancreatic duct in normal children and children with pancreatitis.

We investigated the diameter of pancreatic duct using ultrasonography in 51 children with pancreatitis and age-matched healthy control children over a 5 year period. The diameters of pancreatic duct and pancreatic body were measured simultaneously by sonography. The mean ages of children with acute pancreatitis and chronic pancreatitis were 9.7 +/- 3.9 and 10.3 +/- 3.1 years, respectively (range, 1 to 8 years). The mean age of normal children was 9.6 +/- 5.3 years. A significant difference was found in diameter of the pancreatic duct between children with acute and chronic pancreatitis versus that of age-matched control. In addition, a significant difference in diameter of the pancreatic body was found between children with acute pancreatitis and age-matched controls, but there was no marked difference in diameter of the pancreatic body between normal persons and those with chronic pancreatitis. The mean diameters of the pancreatic duct in acute pancreatitis and chronic pancreatitis were 2.34 +/- 0.47 mm and 2.84 +/- 0.67 mm, respectively, which was greater than that of normal children (1.65 +/- 0.45 mm). Pancreatic ducts with diameters greater than 1.5 mm in children between 1 and 6 years, greater than 1.9 mm at ages 7 to 12 years, or greater than 2.2 mm at ages 13 to 18 years were significantly associated with the presence of acute pancreatitis. Thirty-two patients, including 25 with acute pancreatitis and 7 with chronic pancreatitis, underwent follow-up measurement of pancreatic duct and serum lipase examination on at least three occasions. A good correlation between the diameter of pancreatic duct and serum lipase level was found. Thus, ultrasonography of the pancreatic duct is valuable in diagnosis and monitoring of pancreatitis in children.

Prevalence of mutations associated with reduced antiretroviral drug susceptibility among human immunodeficiency virus type 1 seroconverters in the United States, 1993-1998.

To assess the prevalence of mutations associated with decreased antiretroviral drug susceptibility, specimens were tested from persons infected with human immunodeficiency virus (HIV) during 1993-1998. Subjects were drug naive and were attending sexually transmitted disease clinics in 6 US cities. All were enrolled consecutively and had tested negative for HIV during the 2 years before enrollment. Plasma specimens from patients having >/=1 reverse transcriptase (RT) or primary protease mutation were tested phenotypically with a recombinant virus assay. Of 99 patients, 6 (6%) had mutations associated with zidovudine resistance, 2 (2%) had mutations associated with nonnucleoside RT inhibitor resistance, and 1 (1%) had a primary protease mutation. Overall, the prevalence of resistance-associated primary mutations was 5%, although high levels of decreased drug susceptibility (IC(50)s >/=10 times that of a reference virus) were observed in just 1%. These findings confirm the transmission of these mutations to drug-naive persons.

Distribution of HIV-1 subtypes among HIV-seropositive patients in the interior of Côte d'Ivoire.

Limited data exist on the distribution of HIV-1 subtypes in Côte d'Ivoire. The aim of this study is to describe the distribution of genetic subtypes of HIV-1 strains in six regions of Côte d'Ivoire. In 1997, we consecutively collected blood from 172 HIV-1-infected patients from six regional tuberculosis treatment centers. Peripheral blood mononuclear cells (PBMCs) from these people were analyzed by a restriction fragment-length polymorphism (RFLP) assay that involves a sequential endonuclease digestion of a 297-base pair polymerase chain reaction (PCR) fragment; plasma samples were tested by a V3-loop peptide enzyme immunoassay (PEIA). DNA sequencing of the protease or env genes was performed on all samples discordant in the two assays as well as a random sample of the concordant subtyped samples. Of 172 specimens, 3 were PCR-negative, and 169 were putatively classified as subtype A by RFLP. The 3 PCR-negative samples were unequivocally subtyped A by PEIA. Of the 169 RFLP subtype A samples, 159 (94%) were subtyped A by PEIA. Of the 10 discordant samples, PEIA testing classified 3 as subtype C, 2 as D, and 5 as F. Sequencing of the env gene classified these samples as 1 subtype A, 4 Ds, and 5 Gs. Thus, 163 (95%) of the specimens were subtype A, 3 subtype D, 4 subtype G, 1 A/D, and 1 A/G (IbNG) circulating recombinant forms (CRF). In conclusion, most HIV-1-infected tuberculosis patients throughout the interior of Côte d'Ivoire are infected with HIV-1 subtype A, which are very likely the A/G (IbNG) CRF. The uniform distribution of this subtype makes Côte d'Ivoire a potential site for vaccine trials.

Genetic characterization of incident HIV type 1 subtype E and B strains from a prospective cohort of injecting drug users in Bangkok, Thailand.

We obtained specimens from 128 HIV-1 seroconverters identified from 1995 through 1998 in a prospective cohort study of 1,209 HIV-negative injecting drug users (IDUs) in Bangkok, Thailand. Epidemiologic data indicated that parenteral transmission accounted for nearly all infections. HIV-1 DNA from the C2-V4 env region was sequenced, and phylogenetic analyses determined that 102 (79.7%) of the specimens were subtype E and 26 (20.3%) subtype B strains. All subtype B strains clustered with strains often referred to in previous studies as Thai B or B'. The interstrain nucleotide distance (C2-V4) within subtype E strains was low (mean, 6.8%), and pairwise comparisons with a prototype subtype E strain, CM244, showed limited divergence (mean, 5.6%). The subtype B stains showed greater interstrain divergence (mean, 9.2%) and were significantly divergent from the prototype B strain HIV-MN (mean, 13.0%; p < 0.0001). The subtype E strains had significantly lower mean V3 loop charge than did subtype B strains (p = 0.017) and, on the basis of analysis of amino acid sequences, were predicted to be predominantly (91%) non-syncytium-inducing (NSI), chemokine coreceptor CCR5-using (CCR5+) viruses. The subtype B strains had a higher mean V3 loop charge, and a smaller proportion (23%) were predicted to be NSI/CCR5+ viruses. This study demonstrates that most incident HIV1 infections among Bangkok IDUs are due to subtype E viruses, with a narrow spectrum of genetic diversity. The characterization of incident HIV-1 strains from 1995 to 1998 will provide important baseline information for comparison with any breakthrough infections that occur among IDUs in Bangkok who are participating in an HIV-1 vaccine efficacy trial initiated in 1999.

Repair of hypospadias complications using the tubularized, incised plate urethroplasty.

BACKGROUND/PURPOSE: Secondary procedures to correct complications after hypospadias repair remain challenging especially for "hypospadias cripples." The tubularized, incised plate urethroplasty was first introduced by Snodgrass for the repair of primary hypospadias in 1993. The authors used this procedure to correct the complications after hypospadias repair in patients who had no abundant local skin flaps to be used for a neourethra. METHODS: Six patients underwent tubularized, incised plate urethroplasty for the correction of complications of hypospadias repair performed the previous year, including a large urethrocutaneous fistula (n = 1) and disruption of the neourethra (n = 5). Prior surgical procedures included transverse island tube urethroplasty in 4 cases and 2-stage urethroplasty in 2 cases. The average patient age at the time of secondary procedure was 4.6 years (range, 1 to 12 years). RESULTS: The mean follow-up period was 6 months (range, 2 months to 1 year). All the patients obtained a functional neourethra with a vertical, slitlike meatus. A small fistula developed in one child and mild meatal retraction in another. CONCLUSIONS: The tubularized, incised plate urethroplasty offers few complications and good cosmetic results. The authors recommend its use for patients who have had repeated surgeries for hypospadias repair, especially those in whom only limited local skin flaps can be utilized for a neourethra.

Genetic analysis of human immunodeficiency virus in Abidjan, Ivory Coast reveals predominance of HIV type 1 subtype A and introduction of subtype G.

To better understand the molecular epidemiology of HIV genetic diversity in Abidjan, Ivory Coast, we performed a genetic analysis of 170 HIV-1-seropositive specimens representing newly diagnosed tuberculosis patients (n = 143) and women monitored in a mother-to-child transmission cohort study (n = 27). Preliminary screening with RFLP presumptively classified 162 (95.3%) of these as subtype A. The envelope region of 108 specimens was subtyped by sequence analysis: 102 (94.4%) were subtype A, 2 (1.9%) were subtype D, and 4 (3.7%) were subtype G. Subtyping gag and env regions of the genome suggested that five of the six nonsubtype A isolates exhibited a potentially mosaic structure. A comparative phylogenetic analysis of HIV-1 subtype A C2V3 from 27 Ivory Coast and 21 Ugandan sequences revealed a striking clustering among Ivory Coast variants, and an independent segregation from Ugandan subtype A. Despite independent clustering with other subtype A specimens, limited variability of the V3 loop apex was observed; the globally predominant V3 motif, GPGQ, represented 90.1% of the HIV-1 strains. This study demonstrates that clade A is the predominant HIV-1 subtype in HIV-seropositive individuals in Abidjan, Ivory Coast and that these strains are phylogenetically distinct from other subtype A strains observed in East Africa.

The development and evaluation of a probe hybridization method for subtyping HIV type 1 infection in Uganda.

We developed a method for large-scale screening of HIV-1 genotypic variation based on DNA probe hybridization. Nested PCR amplifications were performed to generate fragments in the env C2-V3 region and also in the gp41 region, which encompasses the immunodominant domain. The proviral DNA sequences were derived from 68 samples and phylogenetically analyzed. For comparison, the C2-V3 fragment was used in DNA probe hybridization to rapidly determine the infecting HIV subtype. The hybridizing probes were designed on the basis of the two most prevalent subtypes in Uganda, A and D. The results were compared to evaluate the feasibility of using this hybridization method for large-scale genotypic screening. Sequence analysis of the 68 amplified PCR fragments showed that 39 were subtype A and 29 were subtype D. The results of DNA hybridization to the amplified products with A and D subtype-specific probes were more than 90% concordant with the subtypes determined by sequence analysis. Our findings suggest that probe hybridization with subtype-specific probes is effective for large-scale screening of HIV-infected populations. Application of this method will significantly reduce the time needed for large, population-based investigations.